PURIFIED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM THERMOTOGA MARITIMA

ENZYME ACIDE NUCLEIQUE POLYMERASE THERMOSTABLE ET PURIFIEE POUR TERMOTOGA MARITIMA

English Abstract

A purified thermostable enzyme is derived from the eubacterium Thermotoga
maritima, The enzyme has a molecular
weight as determined by gel electrophoresis of about 97 kilodaltons and DNA
polymerase I activity. The enzyme can be produced
from native or recombinant host cells and can be used with primers and
nucleoside triphosphates in a temperature-cycling chain
reaction where at least one nucleic acid sequence is amplified in quantity
from an existing sequence.

Claims

-59-

CLAIMS:

1. A thermostable DNA polymerase enzyme having a molecular weight
between about 97 and 103 kilodaltons that catalyzes the combination of
nucleoside triphosphates to form a nucleic acid strand complementary to a
nucleic acid
template strand, wherein said enzyme is derived from the eubacterium
Thermotoga
maritima, has 3' to 5' exonuclease activity and has an optimum temperature at
which it
functions that is higher than about 60°C.
2. The thermostable DNA polymerase enzyme as claimed in claim 1,
which has reverse transcriptase activity.
3. The thermostable DNA polymerase enzyme as claimed in claims 1 or
2, which exhibits activity from about 45°C to 90°C with an
optimum
temperature for activity of 75 to 80°C.
4. The thermostable DNA polymerase enzyme having the amino acid
sequence encoded by the nucleotide sequence with SEQ ID NO: 1, which
amino acid sequence is from amino to carboxy terminus:
Image


-60-



Image
5. A thermostable DNA polymerase enzyme as claimed in any one of claims 1
to 3 substantially identical to the amino acid sequence according to claim 4
or encoded
by a nucleotide sequence substantially identical to the nucleotide sequence
set forth in
SEQ ID NO:1.
6. A thermostable DNA polymerase enzyme having an amino acid sequence
substantially identical to the amino acid sequence of claim 4.
7. An enzymatically active N-terminal shortened fragment of the thermostable
DNA polymerase enzyme as claimed in claim 4, which enzyme has an amino acid
sequence encoded by SEQ ID NO:1, lacking up to 283 amino acids of the amino
terminal sequence.
8. The fragment of claim 7, which has a molecular weight of about 70 or of
about 86 kilodaltons.
9. The fragment of claim 7, which has the amino acid sequence from amino
acids number 140 to 893 or amino acids number 284 to 893 in the amino acid
sequence
encoded by SEQ ID NO:1.
10. The fragment of any one of claims 7 to 9, that lacks 5' to 3' exonuclease
activity or has attenuated 5' to 3' exonuclease activity.
11. The thermostable DNA polymerase enzyme claimed in any one of claims 4
to 6 or the fragment as claimed in any one of claims 7 to 10 which is modified
by
oxidation or reduction, whereby this modification does not destroy the DNA
polymerase
activity or the 3' to 5' exonuclease activity of the resulting thermostable
enzyme.


-61-


12. A modified thermostable DNA polymerase enzyme having 3' to 5'
exonuclease activity and having an amino acid sequence which is modified by
deletion,
addition or alteration relative to an amino acid sequence of a reference
thermostable
DNA polymerase enzyme wherein the modified thermostable DNA polymerase is
encoded by a nucleotide sequence substantially identical to SEQ ID NO:1 which
modified thermostable DNA polymerase retains high temperature DNA polymerase
activity and 3' to 5' exonuclease activity.
13. The thermostable DNA polymerase enzyme as claimed in claim 12, which is
a fusion polypeptide.
14. A chimeric thermostable DNA polymerase enzyme, wherein one or two
domains selected from the group of domains consisting of the 3' .fwdarw. 5'
exonuclease
domain, the 5' .fwdarw. 3' exonuclease domain and the DNA polymerase domain
are from the
polymerase enzyme having the sequence encoded by SEQ ID NO:1 and the other
domain or the other two domains are substituted by codons 423-832 from the Taq
DNA
polymerase or from the Tth DNA polymerase.
15. The thermostable DNA polymerase enzyme of any one of claims 4 to 6, the
fragment as claimed in any one of claims 7 to 10, a modified thermostable DNA
polymerase enzyme as claimed in any one of claims 11 to 13, or a chimeric
polymerase
as claimed in claim 14, that comprises the sequence
D-X-E-X3-L-X55-65-N-X3-D-X3-L-X65-75-Y-X3-D critical for 3' to 5' exonuclease
activity,
whereby X N represents the number (N) of non-critical amino acids between the
specified
amino acids.


-62-


16. A DNA encoding a thermostable DNA polymerase enzyme of any one of
claims 4 to 6, the fragment as claimed in any one of claims 7 to 10, a
modified form of
said thermostable DNA polymerase enzyme as claimed in any one of claims 11 to
13, or
a chimeric polymerase as claimed in claim 14.
17. The DNA having the nucleotide sequence of SEQ ID NO:1:
Image


-63-


Image




-64-
18. A DNA fragment of the thermostable DNA polymerase enzyme from the
eubacterium Thermotoga maritima, which coding sequence has the nucleotide
sequence
of SEQ ID NO:1, lacking up to 849 nucleotides from the 5' end.
19. The DNA sequence of claim 18 having the nucleotide sequence from
nucleotide number 418 to 2682 or from nucleotide number 850 to 2682 of SEQ ID
NO:1.
20. A DNA vector that comprises a DNA sequence as claimed in any one of
claims 16 to 19.
21. A recombinant host cell transformed with a DNA vector as claimed in claim
20, which host cell is a strain of E. coli.
22. A method for purifying a thermostable DNA polymerase enzyme as claimed
in any one of claims 1 to 6 or the fragment as claimed in any one of claims 7
to 10, said
method comprising:
(a) preparing a crude cell extract from cells comprising said polymerase;
(b) adjusting the ionic strength of said extract so that said polymerase
dissociates
from any nucleic acid in said extract;
(c) subjecting the extract to hydrophobic interaction chromatography;
(d) subjecting the extract to DNA binding protein affinity chromatography;
(e) subjecting the extract to nucleotide binding protein affinity
chromatography;
and
(f) subjecting the extract to chromatography selected from the group
consisting of
anion exchange, cation exchange, and hydroxyapatite chromatography.
23. A method for the production of a thermostable DNA polymerase enzyme of
any one of claims 4 to 6, a fragment as claimed in any one of claims 7 to 10,
a modified
form thereof as claimed in any one of claims 11 to 13, or a chimeric
polymerase as
claimed in claim 14, said method comprising the culturing of a host cell
according to




-65-
claim 21 and purifying the recombinant DNA polymerase from the medium or from
the
cells.
24. Use of an oligonucleotide probe directed to regions of dissimilarity
between
Thermus aquaticus polymerase and Thermotoga maritima polymerase, which regions
include stretches of four or more contiguous amino acids from any one or more
of the
regions identified by the following amino acid coordinates (numbering is
inclusive): 5-
10, 73-79, 113-119, 134-145, 191-196, 328-340, 348-352, 382-387, 405-414, 467-
470,
495-, 499, 506-512, 555-559, 579-584, 595-599, 650-655, 732-742, 820-825, 850-
856
of the amino acid sequence encoded by SEQ ID NO:1 for retrieving a DNA
encoding a
thermostable DNA polymerase enzyme from a thermostable organism, which DNA
polymerase enzyme has some properties of Thermus aquaticus polymerase and
other
divergent properties typical for Thermotoga maritima polymerase
25. The thermostable DNA polymerase enzyme purified by the method of claim
22 or produced by a method as claimed in claim 23.
26. A composition comprising a thermostable DNA polymerase enzyme
according to any one of claims 1 to 6, a fragment as claimed in any one of
claims 7 to
10, a modified form thereof as claimed in any one of claims 11 to 13, or a
chimeric
polymerase as claimed in claim 14 in a buffer and a stabilizing agent.
27. A composition according to claim 26, wherein said stabilizing agent is a
non-ionic polymeric detergent.
28. Use of a thermostable DNA polymerase enzyme according to any one of
claims 1 to 6, a fragment as claimed in any one of claims 7 to 10, or a
modified form
thereof as claimed in any one of claims 11 to 13, a chimeric polymerase as
claimed in
claim 14, or a composition according to claim 26 or claim 27 for amplifying a
nucleic
acid.




-66-
29. Use of a thermostable DNA polymerase enzyme according to any one of
claims 1 to 6, a fragment as claimed in any one of claims 7 to 10, or a
modified form
thereof as claimed in any one of claims 11 to 13, a chimeric polymerase as
claimed in
claim 14, or a composition according to claim 26 or claim 27 for reverse
transcribing
RNA.
30. A process for amplifying a nucleic acid which process comprises:
(a) heating a duplex of said nucleic acid at a temperature sufficient to yield
single-stranded molecules of said nucleic acid but not to a temperature which
will
irreversibly denature a thermostable DNA polymerase enzyme according to any
one of
claims 1 to 6, a fragment as claimed in any one of claims 7 to 10, or a
modified form
thereof as claimed in any one of claims 11 to 13, a chimeric polymerase as
claimed in
claim 14, or a composition according to claim 26 or claim 27;
(b) cooling products of step (a) to a temperature and for a time sufficient to
promote hybridization of a primer to a single-stranded molecule resulting from
step (a);
(c) maintaining a mixture resulting from step (b) for a temperature and a time
in
the presence of a thermostable DNA polymerase enzyme according to any one of
claims
1 to 6, a fragment as claimed in any one of claims 7 to 10, or a modified form
thereof
as claimed in any one of claims 11 to 13, a chimeric polymerase as claimed in
claim 14,
or a composition according to claim 26 or claim 27 sufficient to synthesize an
extension
product of a primer complementary to a single-stranded molecule resulting from
step
(a); and
(d) repeating steps (a) to (c) sufficiently often to yield an amplified amount
of
said nucleic acid.
31. A process for reverse transcribing an RNA which process comprises:
incubating an annealed primer and said RNA with a thermostable DNA
polymerase enzyme according to any one of claims 1 to 6, a fragment as claimed
in any
one of claims 7 to 10, or a modified form thereof as claimed in any one of
claims 11 to
13, a chimeric polymerase as claimed in claim 14, or a composition according
to claim
26 or claim 27 under conditions sufficient for said polymerase to catalyze
formation of a
DNA sequence complementary to a sequence of said RNA.

Description

CA 02089495 2001-02-05
PURIFIED THERMGSTABLE NUCLEIC AC)D POLYMERASE
ENZYME FROM THERMOTOGA MAR)TTMA
Technical Field
The present invention relates to a purified, thermostable DNA polymerise
ptuified from the hypertherntophilic eubacteria Thermotoea maritime and means
for
isolating and producing the enzyme. Thermostable DNA polymerises are useful in
many recombinant DNA techniques, especially nucleic acid amplification by the
polymerise chain reaction (PCR).
Background Art
:l0 In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation of the
bacterium Thermotoaa maritime is described. T. maritime is a eubacterium that
is
strictly anaerobic, rod-shaped, fermentative, hyperthermophilic, and grows
between
55'C and 90'C, with an optimum growth temperature of about 80'C. This
eubacterium
has been isolated from geothetmally heated sea floors in Italy and the Azores.
~.
'.5 maritime cells have a sheath-like structure and monotrichous flagellation.
T. maritime
is classified in the eubacterial kingdom by virtue of having murein and fatty
acid-
containing lipids, diphtheria-toxin-resistant elongation factor 2, an RNA
polymerise
subunit pattern, and sensitivity to antibiotics.
Extensive research his bY.en conducted on the isolation of DNA polymerises
a!0 from mesophilic microorganisms such as ~. ~l_i. See, for example, Bessman
gl ~.,
1957, ,~. i 1. Chem. x:171-177, and Buttin and Kornberg, 1966, ~. Biol. h m.
241:5419-5427. Much less investigation has been made on the isolation and
purification of DNA polymerises Trom thermophiles such as Thermotoia maritime.
In
Kaledin et al., 1980, Biokhymiv~~ 45:644-651, a six-step isolation and
enrichment
~:5 procedure for DNA polymerise activity from cells of a Thermus aquaticus YT-
1 strain
is disclosed. These steps involve: isolation of crude extract, DEAF-cellulose
chromatography, fractionation on hydroxyapatite, fractionation on DEAF-
cellulose, and
chromatography on single-strand DNA-cellulose. The molecular weight of the
purified
enzyme is reported by Kaledin e_~, al. as 62,000 daltons per monomeric unit.
30 A second enrichment scheme for a polymerise from Thermus a~uaticus is
described in Chien et al., 1976, ~~. Bacteriol. 127:1550-1557. In this
process, the
crude extract is applied to a DEAF-Sephadex column. The dialyzed pooled
fractions
are then subjected to treatment on a phosphocellulose column. The pooled
fractions are
dialyzed, and bovine serum albumin (BSA) is added to prevent loss of
polymerise
35 activity. The resulting mixture is loaded on a DNA-cellulose column. The
pooled
material from the column is dialyzed. The molecular weight of the purified
protein is
reported to be about 63,000 daltons to 68,000 daltons.
*Trade-mark


CA 02089495 2005-03-08
2
The use of thermostable enzymes, such as those described in Chien gt al. and
Kaledin et a_l., to amplify existing nucleic acid sequences in amounts that
are large
compared to the amount initially present is described in U.S. Patent Nos.
4,683,195;
4,683,202; and 4,965,188, which describe the PCR process. Primers, template,
nucleoside triphosphates, the appropriate buffer and reaction conditions, and
polymerise
are used in the PCR process, which involves denaturation of target DNA,
hybridization of
primers, and synthesis of complementary strands. The extension product of each
primer
becomes a template for the production of the desired nucleic acid sequence.
The patents
disclose that, if the polymerise employed is a thermostable enzyme, then
polymerise need
not be added after every denaturation step, because heat will not destroy the
polymerise
activity.
U.S. Patent No. 4,889,818, European Patent Publication No. 258,017, and PCT
Publication No. 89/06691 describe the isolation and recombinant expression of
an
1~
-94 kDa thermostable DNA polymerise from Thermus aquaticus and the use of that
pvlymerase in PCR. Although ~. aauaticus DNA polymerise is especially
preferred for
use in PCR and other recombinant DNA techniques, there remains a need for
other
thermostable polymerises.
-- 20 Accordingly, there is a desire in the art to produce a purified,
thermostable DNA-
polymerase that may be used to improve the PCR process described above and to
improve the results obtained when using a thermostable DNA polymerise in other
recombinant techniques, such as DNA sequencing, nick-translation, and even
reverse
transcription. The present invention helps meet that need by providing
recombinant
25 expression vectors and purification protocols for ThermotoQa ~ritima DNA
polymerise.
j~isclosure of Invention
The present invention provides a purified thermostable DNA polymerise I
enzyme that catalyzes combination of nucleoside triphosphates to form a
nucleic acid
30 strand complementary to a nucleic acid template strand. The purified enzyme
is the
DNA polymerise I from ~Tnotoea, ~j, 'rte Cue) and has a molecular weight of
about 97 kilodaltons (kDa) as measured by SDS-PAGE and an inferred molecular
weight, from the nucleotide sequence of the ~ DNA polymerise gene, of 102
lcDa.
This purified material may be used in PCR to produce a given nucleic acid
sequence in
35 amounts that are large compared to the amount initially present so that the
sequences
can be manipulated and/or analyzed easily.


CA 02089495 2005-03-08
2a
The invention provides a thermostable DNA polymerise enzyme having a
molecular weight between about 97 and 103 kilodaltons that catalyzes the
combination
of nucleoside triphosphates to form a nucleic acid strand complementary to a
nucleic
acid template strand, wherein said enzyme is derived from the eubacterium
Thermotoga
maritima, has 3' to 5' exonuclease activity and has an optimum temperature at
which it
functions that is higher than about 60°C.
The gene encoding Tma DNA polymerise enzyme from Thermotosa maritima has
also been identified, cloned, sequenced, and expressed at high level and
provides



PCT/US91/05753
WO 92/03556
3
yet another means w prepare the thermostable enzyme of the present invention.
In
addition to the intact gene and the coding sequence for the enzyme,
derivatives of
the coding sequence for DNA polymerise are also provided.
The invention also encompasses a stable enzyme composition comprising a
purified, thermostable ~, enzyme as described above in a buffer containing one
or
more non-ionic polymeric detergents.
Finally, the invention provides a method of purification for the thermostable
polymerise of the invention. This method involves preparing a crude extract
from
fir. .maritima cells, adjusting the ionic strength of the crude extract so
that the
DNA polymerise dissociates from nucleic acid in the extract, subjecting the
extract to
hydrophobic interaction chromatography, subjecting the extract to DNA binding
protein
affinity chromatography, and subjecting the extract to canon or anion exchange
or
hydroxyapatite chromatography. In a preferred embodiment, these steps are
performed
sequentially in the order given above. The nucleotide binding protein affinity
chromatography step is preferred for separating the DNA polymerise from
endonuclease proteins.
Modes for Carrvine out the Invention
The present invention provides DNA sequences and expression vectors that
encode Tma DNA polymerise, purification protocols for Tma DNA polymerise,
preparations of purified ~n DNA polymerise, and methods for using ~ DNA
polymerise. To facilitate understanding of the invention, a number of terms
are defined
below.
The terms "cell," "cell line," and "cell culture" are used interchangeably and
all
such designations include progeny. Thus, the words "transformants" or
"transformed
cells" include the primary transformed cell and cultures derived from that
cell without
regard to the number of transfers. All progeny may not be precisely identical
in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny that have
the same
functionality as screened for in the originally transformed cell are included
in the
definition of transformants.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked coding sequence in a particular bast
organism. The
control sequences that are suitable for procaryotes, for example, include a
promoter,
optionally an operator sequence, a ribosome binding site, and possibly other
sequences, such as transcription termination sequences. Eucaryotic cells are
known to
utilize promoters, polyadenylation signals, and enhancers.
The term "expression system" refers to DNA sequences containing a desired
coding sequence and control sequences in operable linkage, so that hosts
transformed
with these sequences are capable of producing the encoded proteins. To effect


CA 02089495 2001-02-05
4
transformation, the expression system may be included on a vector; the
relevant DNA
can also be integrated into the host chromosome.
The term "gene" refers. to a DNA sequence that codes for the expression of a
recoverable bioactive polypeptide or precursor. Thus, the Tma DNA polymerise
gene
includes the promoter and Tma DNA polymerise coding sequence. The polypeptide
can be encoded by a full-length coding sequence or by any portion of the
coding
sequence so long as the desired enzymatic activity is retained.
The term "operably lir~l:ed" refers to the positioning of the coding sequence
such that control sequences will function to drive expression of the encoded
protein.
Thus, a coding sequence "operably linked" to a control sequence refers to a
configuration wherein the coding sequence can be expressed under the direction
of the
control sequence.
The term "mixture" as it relates to mixtures containing Tma polymerise refers
to
a collection of materials that includes Tma polymerise but can also include
other
proteins. If the Tma polymerise is derived from recombinant host cells, the
other
proteins will ordinarily be those associated with the host. Where the host is
bacterial,
the contaminating proteins will be bacterial proteins.
The term "non-ionic polymeric detergents" refers to surface-active agents that
have no ionic charge and that are characterized, for purposes of this
invention, by an
ability to stabilize the Tma enzyme at a pH range of from about 3.5 to about
9.5,
preferably from 4 to 8.5. Nurnero~us examples of suitable non-ionic polymeric
detergents are presented elsewhere.
The term "oligonucleotide" as used herein is defined as a molecule comprised
of
2~ two or more deoxyribonucleotides or ribonucleotides, preferably more than
three, and
usually more than ten. The exact size will depend on many factors, which in
turn
depend on the ultimate function or use of the oligonucleotide. The
oligonucleotide may
be derived synthetically or by cloning.
The term "primer" as 'used herein refers to an oligonucleotide that is capable
of
~0 acting as a point of initiation of synthesis when placed under conditions
in which
primer extension is initiated. An oligonucleotide "primer" may occur
naturally, as in a
purified restriction digest, or be produced synthetically. Synthesis of a
primer
extension product that is complementary to a nucleic acid strand is initiated
in the
presence of four different nucleoside triphosphates and the Tma thermostable
enzyme in
3~ an appropriate buffer at a suitable temperature. A "buffer" includes
cofactors (such as
divalent metal ions) and salt (to provide tl:e appropriate ionic strength),
adjusted to the
desired pH. For Tma polyme:ras°, the buffer preferably contains 1 to 3
mM of a
magnesium salt, preferably NyCI~, 50 to 200 ~tM of each nucleoside
triphosphate, and
0.2 to 1 ~tM of each primer. along with ~0 mM KC1, 10 mM Tris buffer (pH 8.0-
8.4j,




W~ 92/03556 2 p ~ 9 4 9 ~ PCT/US91/05753
and 100 ~tg/ml gelatin (although gelatin is not required and should be avoided
in some
applications, such as DNA sequencing).
The primer is single-stranded for maximum efficiency in amplification, but may
alternatively be double-stranded. If double-stranded, the primer is first
treated to
5 separate its strands before being used to prepare extension products. The
primer is
usually an oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the
synthesis of extension products in the presence of the polymerise enzyme. The
exact
length of a primer will depend on many factors, such as source of primer and
result
desired, and the reaction temperature must be adjusted depending on primer
length to
ensure proper annealing of primer to template. Depending on the complexity of
the
target sequence, the oligonucleotide primer typically contains 15 to 35
nucleotides.
Short primer molecules generally require cooler temperatures to form
sufficiently stable
complexes with template.
A primer is selected to be "substantially" complementary to a strand of
specific
sequence of the template. A primer must be sufficiently complementary to -
ybridize
with a template strand for primer elongation to occur. A primer sequence need
not
wflect the exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer, with the
remainder of
the primer sequence being substantially complementary to the strand. Non-
compleme ary bases or longer sequences can be interspersed into the primer,
provided
that the primer sequence has sufficient complementarity with the sequence of
the
template to hybridize and thereby form a template/primer complex for synthesis
of the
extension product of the primer.
The terms "restriction endonucleases" and "restriction enzymes" refer to
bacterial enzymes that cut double-stranded DNA at or near a specific
nucleotide
sequence.
The term "therrnostable enzyme" refers to an enzyme which is stable to heat
and
is heat resistant and catalyzes (facilitates) combination of the nucleotides
in the proper
manner to form primer extension products that are complementary to a nucleic
acid
strand. Generally, synthesis of a primer extension product begins at the 3'
end of the
primer and proceeds towards the 5' end of the template strand until synthesis
terminates. A thermostable enzyme must be able to renature and regain activity
after
brief (i.e., 5 to 30 seconds) exposure to temperatures of 80°C to
105°C and must have
a temperature optimum of above 60°C.
The ~ thermostable DNA polymerise enzyme of the present invention
satisfies the requirements for effective use in the amplification reaction
known as the
polymerise chain reaction or PCR. The Tma DNA polymerise enzyme does not
become irreversibly denatured (inactivated) when subjected to the elevated
temperatures
for the time necessary to effect denaturation of double-stranded nucleic
acids, a key step



~ 92/03556 PCT/US91/05753
. ,,.. 2 0 8 9 4 9 5 _
6
in the PCR process. Irreversible denaturation of an enzyme for purposes herein
refers
to permanent and complete loss of enzymatic activity.
The hearing conditions necessary to .effect nucleic acid denaturation will
depend,
e.g., on the buffer salt concentration and the composition, length, and amount
of the
S nucleic acids being denatured, but typically the denaturation temperature
ranges from
about 80'C to about 105'C for a few seconds to minutes. Higher temperatures
may be
required for nucleic acid denaturation as the buffer salt concentration and/or
GC
composition of the nucleic acid is increased. The Tma enzyme does not become
irreversibly denatured upon relatively short exposures to temperatures of
about 80'C-
lOS'C.
The ~ thermostable enzyme has an optimum tempezature at which it
functions that is higher than about 60'C. Temperatures below 60'C facilitate
hybridization of primer to template, but depending on salt composition and
concentration and primer composition and length, hybridization of primer to
template
I5 can occur at higher temperatures (e.g., 60'G-80'C), which may promote
specificity of
the primer elongation reaction. The higher the temperature optimum for the
enzyme,
the greater the specificity and/or selectivity of the primer-directed
extension process.
The ~ enzyme exhibits activity over a broad temperature range from about 45'C
to
90'C; a preferred optimum temperature is 7S'C-80'C.
-- - 20 The present invention also provides DNA sequences encoding the
thermostable
DNA polymerise I activity of ~ermotoQa gtaritima. The amino acid sequence
encoded
by this sequence has homology to portions of the thermostable DNA polymerises
of
Thermus ~ and Thermus thermoRhilus. The complete coding sequence, from
the S'-ATG start codon to the TGA-3' stop codon, of the Tma DNA polymerise
gene is
2S depicted below and listed as SEQ ID NO: 1. The sequence is numbered for
reference.
1 ATGGCGAGAC TATTTCTCTT TGATGGAACT GCTCTGGCCT ACAGAGCGTA
51 CTATGCGCTC GATAGATCGC TTTCTACTTC CACCGGCATT CCCACAAACG
101 CCACATACGG TGTGGCGAGG ATGCTGGTGA GATTCATCAA AGACCATATC
3O 151 ATTGTCGGAA AAGACTACGT TGCTGTGGCT TTCGACAAAA AAGCTGCCAC
201 CTTCAGACAC AAGCTCCTCG AGACTTACAA GGCTCAAAGA CCAAAGACTC
251 CGGATCTCCT GATTCAGCAG CTTCCGTACA TAAAGAAGCT GGTCGAAGCC
301 CTTGGAATGA AAGTGCTGGA GGTAGAAGGA TACGAAGCGG ACGATATAAT
351 TGCCACTCTG GGTGTGAAGG GGCTTCCGCT TTTTGATGAA ATATTCATAG
3S 401 TGACCGGAGA TAAAGACATG CTTCAGCTTG TGAACGAAAA GATCAAGGTG
951 TGGCGAATCG TAAAAGGGAT ATCCGATCTG GAACTTTACG ATGCGCAGAA
501 GGTGAAGGAA AAATACGGTG TTGAACCCCA GCAGATCCCG GATCTTCTGG
551 CTCTAACCGG AGATGAAATA GACAACATCC CCGGTGTAAC TGGGATAGGT
601 GAAA~.GACTG CTGTTCAGCT TCTAGAGAAG TACAAAGACC TCGAAGACAT
B




W~ O 92/03556 '~ ~ ~ ~ ~ ~ ~ PGT/U891/05753
7
651 ACTGAATCATGTTCGCGAACTTCCTCAAAAGGTGAGAAAAGCCCTGCTTC


701 GAGACAGAGAAAACGCCATTCTCAGCAAAAAGCTGGCGATTCTGGAAACA


751 AACGTTCCCATTGAAATAAACTGGGAAGAACTTCGCTACCAGGGCTACGA


801 CAGAGAGAAACTCTTACCACTTTTGAAAGAACTGGAATTCGCATCCATCA


S 851 TGAAGGAACTTCAACTGTACGAAGAGTCCGAACCCGTTGGATACAGAATA


901 GTGAAAGACCTAGTGGAATTTGAAAAACTCATAGAGAAACTGAGAGAATC


951 CCCTTCGTTCGCCATAGATCTTGAGACGTCTTCCCTCGATC :TTCGACT


1001 GCGACATTGTCGGTATCTCTGTGTCTTTCAAACCAAAGGAAGCGTACTAC


1051 ATACCACTCCATCATAGAAACGCCCAGAACCTGGACGAAAAAGAGGTTCT


lO 1101 GAAAAAGCTCAAAGAAATTCTGGAGGACCCCGGAGCAAAGATCGTTGGTC


1151 AGAATTTGAAATTCGATTACAAGGTGTTGATGGTGAAGGGTGTTGAACCT


1201 GTTCCTCCTTACTTCGACACGATGATAGCGGCTTACCTTCTTGAGCCGAA


1251 CGAAAAGAAGTTCAATCTGGAGGATCTCGCATTGAAATTTCTTGGATACA


1301 AAATGACATCTTACCAAGAGCTCATGTCCTTCTCTTTTCCGCTGTTTGGT


IS 1351 TTCAGTTTTGCCGATGTTCCTGTAGAAAAAGCAGCGAACTACTCCTGTGA


1401 AGATGCAGACATCACCTACAGACTTTACAAGACCCTGAGCTTAAAACTCC


1451 ACGAGGCAGATCTGGAAAACGTGTTCTACAAGATAGAAATGCCCCTTGTG


1501 AACGTGCTTGCACGGATGGAACTGAACGGTGTGTATGTGGACACAGAGTT


1551 CCTGAAGAAACTCTCAGAAGAGTACGGAAAAAAACTCGAAGAACTGGCAG


ZO 1601 AGGAAATATACAGGATAGCTGGAGAGCCGTTCAACATAAACTCACCGAAG


1651 CAGGTTTCAAGGATCCTTTTTGAAAAACTCGGCATAAAACCACGTGGTAA


1701 AACGACGAAAACGGGAGACTATTCAACACGCATAGAAGTCCTCGAGGAAC


1751 TTGCCGGTGAACACGAAATCATTCCTCTGATTCTTGAATACAGAAAGATA


1801 CAGAAATTGAAATCAACCTACATAGACGCTCTTCCCAAGATGGTCAACCC


?,S 1851 AAAGACCGGAAGGATTCATGCTTCTTTCAATCAAACGGGGACTGCCACTG


1901 GAAGACTTAGCAGCAGCGATCCCAATCTTCAGAACCTCCCGACGAAAAGT


1951 GAAGAGGGAAAAGAAATCAGGAAAGCGATAGTTCCTCAGGATCCAAACTG


2001 GTGGATCGTCAGTGCCGACTACTCCCAAATAGAACTGAGGATCCTCGCCC


2051 ATCTCAGTGGTGATGAGAATCTTTTGAGGGCATTCGAAGAGGGCATCGAC


3O 2101 GTCCACACTCTAACAGCTTCCAGAATATTCAACGTGAAACCCGAAGAAGT


2151 AACCGAAGAAATGCGCCGCGCTGGTAAAATGGTTAATTTTTCCATCATAT


2201 ACGGTGTAACACCTTACGGTCTGTCTGTGAGGCTTGGAGTACCTGTGAAA


2251 GAAGCAGAAAAGATGATCGTCAACTACTTCGTCCTCTACCCAAAGGTGCG


2301 CGATTACATTCAGAGGGTCGTATCGGAAGCGAAAGAAAAAGGCTATGTTA


3S 2351 GAACGCTGTTTGGAAGAAAAAGAGACATACCACAGCTCATGGCCCGGGAC


2401 AGGAACACACAGGCTGAAGGAGAACGAATTGCCATAAACACTCCCATACA


2451 GGGTACAGCAGCGGATATAATAAAGCTGGCTATGATAGAAATAGACAGGG


2501 AACTGAAAGAAAGAAAAATGAGATCGAAGATGATCATACAGGTCCACGAC


2551 GAACTGGTTTTTGAAGTGCCCAATGAGGAAAAGGACGCGCTCGTCGAGCT






W0~2/03556 2 0 8 9 4 9 5 _ PCT/US91/05753
8
601 GGTGAAAGAC AGAATGACGA ATGTGGTAAA GCTTTCAGTG CCGCTCGAAG
2651 TGGATGTAAC
CATCGGCAAA ACaTGGTCGT
Ga


Both the complete ing sequence of the
cod Tma DNA polymerise
gcnc and the encoded


amino acid sequence
in three letter
abbreviation are
provided. For
convenience, the
amino


acid sequence encoded
by the Tma DNA
polymerise gene
sequence is also
depicted below


in one letter abbreviation
from amino-terminus
to carboxy-terminus;
the sequence is


numbered for reference.


1 MARLFLFDGT ALAYRAYYAL DRSLSTSTGI PTNATYGVAR MLVRFIKDHI


51 IVGKDYVAVA FDKKAATFRH KLLETYKAQR PKTPDLLIQQ LPYIKKLVEA


101 LGMKVLEVEG YEADDIIATL AVKGLPLFDE IFIVTGDKDM LQLVNEKIKV


151 WRIVKGISDL ELYDAQKVKE KYGVEPQQIP DLLALTGDEI DNIPGVTGIG


201 EKTAVQLLEK YKDLEDILNH VRELPQKVRK ALLRDRENAI LSKKLAILET


251 NVPIEINWEE LRYQGYDREK LLPLLKELEF ASIMKELQLY EESEPVGYRI


301 VKDLVEFEKL IEKLRESPSF AIDLETSSLD PFDCDIVGIS VSFKPKEAYY --


351 IPLHHRNAQN LDEKEVLKKL KEILEDPGAK IVGQNLKFDY KVLMVKGVEP


40i VPPYFDTMIA AYLLEPNEKK FNLDDLALKF LGYKMTSYQE LMSFSFPLFG


451 FSFADVPVEK AANYSCEDAD ITYRLYKTLS LKLHEADLEN VFYKIEMPLV


501 NVLARMELNG VYVDTEFLKK LSEEYGKKLE ~LAEEIYRIA GEPFNINSPK


551 QVSRILFEKL GIKPRGKTTK TGDYSTRIEV LEELAGEHEI IPLILEYRKI


v01 QKLKSTYIDA LPKMVNPKTG RIHASFNQTG TATGRLSSSD PNLQNLPTKS


651 EEGKEIRKAI VPQDPNWWIV SADYSQIELR ILAHLSGDEN LyRAFEEGID


701 VHTLTASRIF NVKPEEVTEE MRRAGKMVNF SIIYGVTPYG LSVRLGVPVK


751 EAEKMIVNYF VLYPKVRDYI QRWSEAKEK GYVRTLFGRK RDIPQLMARD


801 RNTQAEGERI AINTPIQGTA ADIIKLAMIE IDRELKERKM RSKMIIQVHD


851 ELVFEVPNEE KDALVELVKD RMTNVVKLSV PLEVDVTIGK TWS


The one letter
abbreviations
for the amino
acids are shown
below for convenience.


F - Phenylalanine H - Histidine


L - Leucine Q - Glutamine


I - Isoleucine N - Asparagine


M - Methionine K - Lysine


V - Valine D - Aspartic Acid


S - Serine E - Glutamic Acid


P - Proline C - Cysteine


T - Threonine W - Tryptophan


A - Alanine R _ Arginine


Y - Tyrosine G - Glycine


The coding sequ ence for Tma DNA polymerise
I was identified by
a


"dcgenerate primer"
method that has
broad utility
and is an important
aspcct of the


present invention.degenerate primer method,
In the DNA fragments of any



B




WO 92/03556 PCT/US91/05753
9
thermostable polymerise coding sequence corresponding to conserved domains of
known thetmostable DNA polymerises can be identified.
In one embodiment of the degenerate primer method, the corresponding
conserved domains are from the coding sequences for and amino acid sequences
of the
thermostable DNA polymerises of ~, ~, and ~. The degenerate primer method
was developed by comparing the amino acid sequences of DNA polymerise I
proteins
from ~,q,, ~, T7, and ~. ~ in which various conserved regions were identified.
Primers corresponding to these conserved regions were then designed. As a
result of
the present invention, sequences can be used to design other degenerate
primers.
The generic utility of the degenerate primer process is exemplified herein by
specific
reference to the method as applied to cloning the gene.
To clone the r~ DNA polymerise I gene, the conserved amino acid sequences
were converted to all of the possible colons for each of the amino acids. Due
to the
degenerate nature of the genetic code, a given amino acid may be represented
by several
different colons. Where more than one base can be present in a colon for a
given
amino acid, the sequence is said to be degenerate.
The primers were then synthesized as a pool of all of the possible DNA
sequences that could code for a given amino acid sequence. The amount of
degeneracy
of a given primer pool can be determined by multiplying the number of possible
nucleotides at each position.
The more degenerate a primer pool, (i.e., the greater the number of individual
unique primer DNA sequences within the pool), the greater the probability that
one of
the unique primer sequences will bind to regions of the target chromosomal DNA
other
than the one desired -- hence, the lesser the specificity of the resulting
amplification.
To increase the specificity of the amplification using the degenerate primers,
the pools
are synthesized is subsets such that the entire group of subsets includes all
possible
DNA sequences encoding the given amino acid sequence, but each individual
subset
only includes a portion: for example, one pool may contain either a G or C it
a
particular position while the other contains either an A or T at the same
position. Each
of these subpools is designated with a DG number.
Both forward primers (directed from the 5' region toward the 3' region of the
gene, complementary to the noncoding strand) and reverse primers (directed
from the
3' region toward the 5' region of the gene, complementary to the coding
strand) were
designed for most of these conserved regions to clone polymerise. The primers
were designed with restriction sites at the 5' ends to facilitate cloning. The
forward
primers contained a III restriction site (AGATCT), while the reverse primers
contained an SRI restriction site (GAATTC). In addition, the primers contained
2
nucleotides at the S' end to increase the efficiency of cutting at the
restriction site.




WO 92/03556 ~ ~ ~ ~ ~ ~ ~ PCT/US91/05753
Degenerate primers were then used in PCR processes in which the target nucleic
acid was chromosomal DNA from Thermotoga maritima. The products of the PCR
processes using a combination of forward and reverse primer pools in
conjunction with
a series of temperature profiles were compared. When specific products of
similar size
5 to the product generated using ~ chromosomal DNA were produced, the PCR
fragments were gel purified, reamplified and cloned into the vector
BSM13H3:BgIII (a
derivative of the Stratagene vector pBSM+ in which the Hin site of pBSM+ was
converted to a Bgl_II site). Sequences were identified as potential
thetmostable DNA
polymerise coding sequences if the sequences were found to encode amino acid
10 sequences homologous to other known amino acid sequences in polymerise
proteins,
particularly those of ~ polymerise and ~1 polymerise.
The portions of the Tma DNA polymerise gene were then identified in the
chromosomal DNA of Thermotoea maritima by Southern blot analysis. The Tma
chromosomal DNA was digested with a variety of enzymes and transferred to
nitrocellulose filters. Probes labeled with 32P or biotin-dUTP were generated
for
various regions of the gene from the cloned PCR products. The probes were
hybridized to the nitrocellulose-bound genomic DNA, allowing identification of
the size
of the chromosomal DNA fragment hybridizing to the probe. The use of probes
covering the 5' and 3' regions of the gene ensures that the DNA fragments)
contain
most if not all of the structural gene for the polymerise. Restriction enzymes
are
identified that can be used to produce fragments that contain the structural
gene in a
single DNA fragment or in several DNA fragments to facilitate cloning.
Once identified, the chromosomal DNA fragments encoding the ~,a DNA
polymerise gene were cloned. Chromosomal DNA was digested with the identified
restriction enzyme and size fractionated. Fractions containing the desired
size range
were concentrated, desalted, and cloned into the BSM13H3:BgIII cloning vector.
Clones were identified by hybridization using labeled probes generated from
the
previous cloned PCR products. The PCR products were then analyzed on
polyacrylamide gels.
The DNA sequence and amino acid sequence shown above and the DNA
compounds that encode those sequences can be used to design and construct
recombinant DNA expression vectors to drive expression of Tma DNA polymerise
activity in a wide variety of host cells: A DNA compound encoding all or part
of the
DNA sequence shown above can also be used as a probe to identify thermostable
polymerise-encoding DNA from other organisms, and the amino acid sequence
shown
above can be used to design peptides for use as immunogens to prepare
antibodies that
can be used to identify and purify a thermostable polymerise.
Whether produced by recombinant vectors that encode the above amino acid
sequence or by native Thermotosa maritima cells, however, Tma DNA polymerise
will




W~2/03556 ~ ~ ~ PCT/US91/05753
11
typically be purified prior to use in a recombinant DNA technique. The present
invention provides such purification methodology.
For recovering the native protein, the cells are grown using any suitable
technique. Briefly, the cells are grown in "MMS"-medium containing (per
liter): NaCI
(6.93 g); MgS04-7H20 ( 1.75 g); MgCl2-6H20 ( 1.38 g); KCl (0.16 g); NaBr (25
mg);
H3B03 (7.5 mg); SrC12,6H20 (3.8 mg); KI (0.025 mg); CaCl2 (0.38 g); KH2P04
(0.5 g); Na2S (0.5 g); (NH4)2Ni(S04)2 (2 mg); trace minerals (Batch ~ ~1.,
1979,
Mi i 1. Rev. 4:260-296) (15 ml); resazurin (1 mg); and starch (5 g) at a pH of
6.5
(adjusted with H2S04). For growth on solid medium, 0.8% agar (Oxoid) may be
added to the medium. Reasonab:~ growth of the cells also occurs in "SME"-
medium
(Stetter gl ~., 1983, ~I. ~. Microbj~. 4_:535-S51) supplemented with 0.5%
yeast
extract, or in marine broth (Difco 2216).
After cell growth, the isolation and purification of the enzyme takes place in
six
stages, each of which is carried out at a temperature below room temperature,
preferably about 0'C to about 4'C, unless stated otherwise. In the first stage
or step,
the cells, if frozen, are thawed, lysed in an Aminco french pressure cell (8-
20,000 psi),
suspended in a buffer at about pH 7.5, and sonicated to reduce viscosity.
In the second stage, ammonium sulfate is added to the lysate to prevent the
Tma
DNA polymerise from binding to DNA or other cell lysate proteins. Also in the
second_
stage, Polymin l~polyethyleneimine, PEI) is added to the lysate to precipitate
nucleic
acids, and the lysate is centrifuged.
In the third step, ammonium sulfate is added to the su- ~tatant, and the
supernatant is loaded onto a phenyl sepharose column equilibrated with a
buffer
composed of TE (50 mM Tris-Cl, pH 7.5, and 1 mM EDTA) containing 0.3 M
ammonium sulfate and 0.5 mM DTT (dithiothreitol). The column is then washed
first
with the same buffer, second with TE-DTT (without ammonium sulfate), third
with
ethylene glycol-TE-DTT, end finally with 2 M urea in TE-DTT containing
ethylene
glycol. Unless the capacity of the phenylsepharose is exceeded (i.e. by
loading more
than 20-30 mg of protein per ml of resin) all of the ~ polymerise activity is
retained
by the column and elutes with the 2 M urea in TE-DTT containing ethylene
glycol.
In the fourth stage, the urea eluate is applied to a heparin sepharose
colutnrt
which is equilibrated with 0.08 M KCI, 50 mM Tris-CI (pH 7.5), 0.1 mM EDTA,
0.2% Tween 20 a d 0.5 mM DTT. The column is then washed in the same buffer and
the enzyme eluted with a linear gradient of 0.08 M to 0.5 M KCl buffer. The
peak
activity .fractions were found at 0.225 M to 0.275 M KCI.
In the fifth stage, the fraction collected in the fourth stage is diluted with
affigel-
blue buffer without KCl and applied to an affigel-blue column equilibrated in
25 mM
Tris-Cl (pH 7.5), 0.1 mM EDTA, 0.2% Tween 20, 0.5 mM DTT, and 0.15 M KCI.
The column is washed with the same buffer and eluted with a linear gradient of
0.15 M
B




WO 92/03556 PGT/US91/05753~
2~~9495'
12
to 0.7 M KCl in the same buffer. The peak activity fractions were found at the
0.3 M
to 0.55 M KCl section of the gradient. These fractions of peak activity are
then tested
for contaminating deoxyribonucleases (endonucleases and exonucleases) using
any
suitable procedure. As an example, endonuclease activity may be determined
electrophoretically from the change in molecular weight of phage ~, DNA or
supercoiled
plasmid DNA after incubation with an excess of DNA polymerise. Similarly,
exonuclease activity may be determined electrophoretically from the change in
molecular weight of restriction enzyme digested DNA after incubation with an
excess of
DNA polymerise. The fractions that have no deoxyribonuclease activity are
pooled and
diafiltered into phosphocellulose buffer containing 50 mM KCI.
Finally, in a sixth stage, the diafiltered pool from stage five is loaded onto
a
phosphocellulose column equilibrated to the correct pH and ionic strength of
25 mM
Tris-Cl (pH 7.5), 50 mM KCI, 0.1 mM EDTA, 0.2% Tween 20, and 0.5 mM DTT.
The column is then washed with the same buffer and eluted with a linear 0.05 M
to 0.5
M KCl gradient. The peak fractions eluted between 0.215 M and 0.31 M KCI. An
undegraded, purified DNA polymerise from these fractions is evidenced by an
unchanged migration pattern in an ~ ~ activity gel.
The molecular weight of the DNA polymerise purified from Thermotogg
may be determined by any technique, for example, by SDS-PAGE analysis
using protein molecular weight markers or by calculation from the coding
sequence.
The molecular weight of the DNA polymerise purified from ermotogg maritima is
determined by SDS-PAGE to be about 97 kDa. Based on the predicted amino acid
sequence, the molecular weight is estimated at about 102 kDa. The purification
protocol of native Tma DNA polymerise is described in more detail in Example
1.
Purification of the recombinant Tma polymerise of the invention can be canted
out with
similar methodology.
Biologically active recombinant Tma polymerises of various molecular weights
can be prepared by the methods and vectors of the present invention. Even when
the
complete coding sequence of the ~ DNA polymerise gene is present in an
expression vector in _E. coli, the cells produce a truncated polymerise,
formed by
translation starting with the methionine codon at position 140. One can also
use
recombinant means to produce a truncated polymerise corresponding to the
protein
produced by initiating translation at the methionine codon at position 284 of
the
coding sequence. The polymerise lacking amino acids 1 though 139 (about 86
kDa),
and the polymerise lacking amino acids 1 through 283 (about 70 kDa) of the
wild type
Tma polymerise retain polymerise activity but have attenuated 5'--~3'
exonuclease
activity. In addition, the 70 kDa polymerise is significantly more
thermostable than
native Tma polymerise.




WO 92/03556 ~ ~ ~ ~ ~ ~ ~ PGT/US91/05753
13
Thus, the entire sequence of the intact DNA polymerise I enzyme is not
required for activity. Portions of the DNA polymerise I coding sequence can be
used in recombinant DNA techniques to produce a biologically active gene
product with
DNA polymerise activity. The availability of DNA encoding the DNA polymerise
sequence provides the opportunity to modify the coding sequence so as to
generate
mutein (mutant protein) forms also having DNA polymerise activity. The
amino(N)-
terminal portion of the Tma polymea~ase is not necessary for polymerise
activity but
rather encodes the 5'-~3' exonucleasc activity of the protein. Using
recombinant DNA
methodology, one can delete approximately up to one-third of the N-terminal
coding
sequence of the ~ gene, clone, and express a gene product that is quite active
in
polymerise assays but, depending on the extent of the deletion, has no 5'~3'
exonuclease activity. Because certain N-terminal shortened forms of the
polymerise
are active, the gene constructs used for expression of these polymerises can
include the
corresponding shortened forms of the coding sequence.
In addition to the N-terminal deletions, individual amino acid residues in the
peptide chain of ~ polymerise may be modified by oxidation, reduction, or
other
derivation, and the protein may be cleaved to obt: s fragments that retain
activity. Such
alterations that do not destroy activity do not rem::ve the protein from the
definition of a
protein with Tma polymerise activity and so are specifically included within
the scope
of the present invention.
Modifications to the primary structure of the ~n DNA polymerise coding
sequence by deletion, addition, or alteration so as to change the amino acids
incorporated into the ~ DNA polymerise during translation of the mRNA produced
from that coding sequence can be made without destroying the high temperature
DNA
polymerise activity of the protein. Such substitutions or other alterations
result in the
production of proteins having an amino acid sequence encoded by DNA falling
within
the contemplated scope of the present invention. Likewise, the cloned genomic
sequence, or homologous synthetic sequences, of the ~ DNA polymerise gene can
be used to express a fusion polypeptide with ~ DNA polymerise activity or to
express a protein with an amino acid sequence identical to that of native Tma
DNA
polymerise. In addition, such expression can be directed by the Tma DNA
polymerise
gene control sequences or by a control sequence that functions in whatever
host is
chosen to express the Tma DNA polymerise.
Thus, the present invention provides a coding sequence for ~ DNA
polymerise from which expression vectors applicable to a variety of host
systems can
be constructed and the coding sequence expressed. Portions of the polymerase-
encoding sequence are also useful as probes to retrieve other thern~ostable
polymerase-
encoding sequences in a variety of species. Accordingly, oligonucleotide
probes that
encode at least four to six amino acids can be synthesized and used to
retrieve additional



2~~9~9~
WO 9Z/03556 PGT/US91/05753
14
DNAs encoding a thetmostable polymerise. Because there may not be an exact
match
between the nucleotide sequence of the thermostable DNA polymerise gene of
ThermotoQa maritima and the corresponding gone of other species, oligomers
containing approximately 12-18 nucleotides (encoding the four to six amino
sequence)
are usually necessary to obtain hybridization under conditions of sufficient
stringency
to eliminate false positives. Sequences.cncoding six amino acids supply ample
information for such probes. Such oligonucleotide probes can be used as
primers in
the degenerate priming method of the invention to obtain thermostable
polymerise
encoding sequences.
The present invention, by providing coding sequences and amino acid
sequences for Tma DNA polymerise, therefore enables the isolation of other
thermostable polymerise enzymes and the coding sequences for those enzymes.
The
amino acid sequence of the Tma DNA polymerise I protein is very similar to the
amino
acid sequences for the thermostable DNA polymerises of T~ and T~h. These
similarities facilitated the identification and isolation of the Tma DNA
polymerise
coding sequence. The areas of similarity in the coding sequences of these
three
thermostable DNA polymerises can be readily observed by aligning the
sequences.
However, regions of dissimilarity between the coding sequences of the three
thermostable DNA polymerises can also be used as probes to identify other
thermostable polymerise coding sequences that encode thermostable polymerise
enzymes. For example, the coding sequence for a thermostable polymerise having
some properties of ~ and other divergent properties of ~ may be identified by
using probes directed to sequences that encode the regions of dissimilarity
between ~
-wand Tma. Specifically, such regions include a stretch of four or more
contiguous
~3~ amino acids from any one or more of the following regions, identified by
amino acid
sequence coordinates (numbering is inclusive): 5-10, 73-79, 113-119, 134-145,
191-196, 328-340, 348-352, 382-387, 405-414, 467-470, 495-499, 506-512,
555-559, 579-584, 595-599, 650-655, 732-742, 820-825, 850-856. These regions
may be considered as "hallmark motifs" and define additional regions of
critical amino
acid signature sequences for thermostable DNA polymerise functions (e.g. 5'-
~3'
E exonuclease activity, 3'~5' exonuclease activity, and DNA polymerise
activity).
One property found in the Tma DNA polymerise, but lacking in native
DNA polymerise and native T~h DNA polymerise, is 3'-~5' exonuclease activity.
This 3'-~5' exonuclease activity is generally considered to be desirable,
because
misincorporated or unmatched bases of the synthesized nucleic acid sequence
are
eliminated by this activity. Therefore, the fidelity of PCR utilizing a
polymerise with
3'-~5' exonuclease activity (e.g. Tma DNA polymerise) is increased. The 3'~5'
exonuclease activity found in rr DNA polymerise also decreases the probability
of
the formation of primer/dimer complexes in PCR. The 3'~5' exonuclease activity
in




WO 92/03556 ~ p ~ ~ ~ ~ ~ PGT/US91/05753
effect prevents any extra dNTPs from attaching to the 3' end of the primer in
a non-
template dependent fashion by removing any nucleotide that is attached in a
non-
template dependent fashion. The 3'--~5' exonuclease activity can eliminate
single-
stranded DNAs, such as primers or single-stranded template. In essence, every
3'-
5 nucleotide of a single-stranded primer or template is treated by the enryme
as
unmatched and is therefore degraded. To avoid primer degradation in PCR, one
can
add phosphorothioate to the 3' ends of the primers. Phosphorothioate modified
nucleotides are more resistant to removal by 3'-~5' exonucleases.
A "motif' or characteristic "signature sequence" of amino acids critical for
10 3'-~5' exonuclease activity in thercnostable DNA polymerises can ~~e
defincd as
comprising three short domains. Below, these domains are identified as A, B,
and C,
with critical amino acid residues shown in one letter abbreviation and non-
critical
residues identified as "x."
Representative
15 Domain S~uence Tma Coordinates
A DxExxxL 323-329
B NxxxDxxxL 385-393
C YxxxD 464-468
The distance between region A and region B is 55-65 amino acids. The distance
between region B and region C is 67-75 amino acids, preferably about 70 amino
acids.
In ~ DNA polymerise, the amino acids that do not define the critical motif
signature
sequence amino acids are L and TSS, respectively, in domain A; LKF and YKV,
respectively, in domain B; and SCE in domain C. Domain A is therefore DLETSSL;
domain B is NLKFDYKVL; and domain C is YSCED in Tma DNA polymerise I.
Thus, the present invention provides a thenmostable DNA polymerise possessing
3'~5' exonuclease activity that comprises domains A, B, and C, and, more
particularly comprises the sequence D-X-E-X3-L-Xss-bs_N-X3-D_x3-L-X6s-~s-y_X3_
D, where one letter amino acid abbreviation is used, and XN represents
the'number (N)
of non-critical amino acids between the specified amino acids.
- A thermostable 3'-~5' exonuclease domain is represented by amino acids 291
through 484 of Tma DNA polymerise. Accordingly, "domain shuffling" or
construction of "therrnostable chimeric DNA polymerises" may be used to
provide
thermostable DNA polymerises containing novel properties. For example,
substitution
of the DNA polymerise coding sequence comprising colons about 291 through
about 484 for the Thermos ~,aticus DNA polymerise I colons 289-422 would yield
a
novel thermostable DNA polymerise containing the 5'-~3' exonuclease
domain of ~ DNA polymerise ( 1-289), the 3'-~5' exonuclease domain of ~ DNA
polymerise (291-484), and the DNA polymerise domain of T~ DNA polymerise
(423-832). Alternatively, the 5'~3' exonuclease domain and the 3'~5'
exonuclease




WO 92/03556 2, ~ ~ ~ ~ ~ ~ PCT/US91/05753
16
domain of ~ DNA polymerise (ca. codons 1-484) may be fused to the DNA
polymerise (dNTP binding and primer/template binding domains) portions of
DNA polymerise (ca. codons 423-832). The donors and recipients need not be
limited
to ~ and ~n DNA polymerises. ~ DNA polymerise provides analogous domains
as ~ DNA polymerise. In addition, the enhanced/preferred reverse transcriptase
properties of T~h DNA polymerise can be further enhanced by the addition of a
3'~S'
exonuclease domain as illustrated above.
While any of a variety of means may be used to generate chimeric DNA
polymerise coding sequences (possessing novel properties), a preferred method
employs "overlap" PCR. In this method, the intended junction sequence is
designed
into the PCR primers (at their 5'-ends). Following the initial amplification
of the
individual domains, the various products are diluted (ca. 100 to 1000-fold)
and
combined, denatured, annealed, extended, and then the final forward and
reverse
primers are added for an otherwise standard PCR.
Thus, the sequence that codes for the 3'~5' exonuclease activity of Tma DNA
polymerise can be removed from Tma DNA polymerise or added to other
polymerises
that lack this activity by recombinant DNA methodology. One can even replace,
in a
non-thermostable DNA polymerise, the 3'~5' exonuclease activity domain with
the
thermostable 3'-~5' exonuclease domain of Tma polymerise. Likewise, the 3'-~5'
exonuclease activity domain of a non-thermostable DNA polymerise can be used
to
replace the 3'-~5' exonuclease domain of Tma polymerise (or any other
thermostable
polymerise) to create a useful polymerise of the invention. Those of skill in
the art
recognize that the above chimeric polymerises are most easily constructed by
recombinant DNA techniques. Similar chimeric polymerises can be constructed by
moving the 5'~3' exonuclease domain of one DNA polymerise to another.
Whether one desires to produce an enzyme identical to native Tma DNA
polymerise or a derivative or homologue of that enzyme, the production of a
recombinant forni of Tma polymerise typically involves the construction of an
expression vector, the transformation of a host cell with the vector, and
culture of the
transformed host cell under conditions such that expression will occur.
To construct the expression vector, a DNA is obtained that encodes the mature
(used here to include all chimeras or muteins) enzyme or a fusion of the Tma
polymerise to an additional sequence that does not destroy activity or to an
additional
sequence cleavable under controlled conditions (such as treatment with
peptidase) to
give an active protein. The coding sequence is then placed in operable linkage
with
suitable control sequences in an expression vector. The vector can be designed
to
replicate autonomously in the host cell or to integrate into the chromosomal
DNA of the
host cell. The vector is used to transform a suitable host, and the
transformed host is
cultured under conditions suitable for expression of recombinant Tma
polymerise. The




WO 92/03556 2 ~ ~ I~ ~ (~ ~C PGT/US91/05753
17
r~ polymerase is isolated from the w:. :;,hum or from the cells, although
recovery and
purification of the protein may not be necessary in some instances.
Each of the foregoing steps can be done in a variety of ways. For example, the
desired coding sequence may be obtained from genomic fragments and used
directly in
appropriate hosts. The construction for pression vectors operable in a variety
of
hosts is made using appropriate replicons and control sequences, as set forth
generally
below. Construction of suitable vectors containing the desired coding and
control
sequences employs standard ligation and restriction techniques that arc well
understood
in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are
cleaved, modified, and religated in the form desired. Suitable restriction
sites can, if
not normally available, be added to the ends of the coding sequence so as to
facilitate
construction of an expression vector, as exemplified below.
Site-specific DNA cleavage is performed by treating with suitable restriction
enzyme (or enzymes) under conditions that are generally understood in the art
and
specified ~y the manufacturers of commercially available restriction enzymes.
See,
e.g., New England Biolabs, Product Catalog. In gew. -a1, about 1 ~.g of
plasmid or
other DNA is cleaved by one unit of enzyme in about .~0 N.1 of buffer
solution; in the
examples below, an excess of restriction enzyme is generally used to ensure
complete
digestion of the DNA. Incubation times of about one to two hours at about
3'°C are
typical, although variations can be tolerated. After each incubation, protein
i~ ~~emoved
by extraction with phenol and chloroform; this extraction can be followed by
ether
extraction and recovery of the DNA from aqueous fractions by precipitation
with
ethanol. If desired, size separation of the cleaved fragments may be performed
by
polyacrylamide gel or agarose gel electrophoresis using standard techniques.
See, e.g.,
Methods in Enz~molo_gv, 1980, X5:499-560.
Restriction-cleaved fragments with single-strand "overhanging" termini can be
made blunt-ended (double-strand ends) by treating with the large fragment of
~. ~i
DNA polymerase I (Klenow) in the presence of the four deoxynucleoside
triphosphates
(dNTPs) using incubation times of about 15 to 25 minutes at 20°C to
25°C in 50 mM
Tris pH 7.6, 50 mM NaCI, 10 mM MgCl2, 10 mM DTT, and 5 to 10 ItM dN'TPs. The
Klenow fragment fills in at 5' protruding ends, but chews back protruding 3'
single
strands, even though the four dNTPs are present. If desired, selective repair
can be
performed by supplying only one of the, or selected, dNTPs within the
limitations
dictated by the nature of the protruding ends. After treatment with HIenow,
the mixture
is extracted with phenol/chloroform and ethanol precipitated. Similar results
can be
achieved using S 1 nuclease, because treatment under appropriate conditions
with S 1
nuclease results in hydrolysis of any single-stranded portion of a nucleic
acid.
Synthetic oligonucleotides can be prepared using the triester method of
Matteucci ~t ~1., 1981, J. Am. hem. o~. 103:3185-3191, or automated synthesis




WO 92/03556 PGT/US91/05753
2089~9~ is
methods. Kinasing of single strands prior to annealing or for labeling is
achieved using
an excess, e.g., approximately 10 units, of polynucleotide kinase to 0.5 N.M
substrate
in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol (DTT),
and
1 to 2 ~tM ATP. If kinasing is for labeling of probe, the ATP will contain
high specific
activity ~3zP.
Ligations are performed in 15-30 ~t.l volumes under the following standard
conditions and temperatures: 20 mM Tris-Cl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 33
~,g/ml BSA, 10 mM-50 mM NaCI, and either 40 ~.M ATP and 0.01-0.02 (Weiss)
units
T4 DNA ligase at 0'C (for ligation of fragments with complementary single-
stranded
ends) or 1 mM ATP and 0.3-0.6 units T4 DNA ligase at 14'C (for "blunt end"
ligation). Intermolecular ligations of fragments with complementary ends are
usually
performed at 33-100 ~g/ml total DNA concentrations (5 to 100 nM total ends
concentration). Intermolecular blunt end ligations (usually employing a 20 to
30 fold
molar excess of linkers, optionally) are performed at 1 ~tM total ends
concentration.
In vector construction, the vector fragment is commonly treated with bacterial
or
calf intestinal alkaline phosphatase (BAP or CIAP) to remove the 5' phosphate
and
prevent religation and reconstruction of the vector. BAP and CIAP digestion
conditions are well known in the art, and published protocols usually
accompany the
commercially available BAP and CIAP enzymes. To recover the nucleic acid
fragments, the preparation is extracted with phenol-chloroform and ethanol
precipitated
to remove AP and purify the DNA. Alternatively, religation can be prevented by
restriction enzyme digestion of unwanted vector fragments before or after
ligation with
the desired vector.
For portions of vectors or coding sequences that require sequence
modifications, a variety of site-specific primer-directed mutagenesis methods
are
available. The polymerase chain reaction (PCR) can be used to perform site-
specific
mutagenesis. In another technique now standard in the art, a synthetic
oligonucleotide
encoding the desired mutation is used as a primer to direct synthesis of a
complementary nucleic acid sequence of a single-stranded vector, such as pBS
13+, that
serves as a template for construction of the extension product of the
mutagenizing
primer. The mutagenized DNA is transformed into a host bacterium, and cultures
of
the transformed bacteria are plated and identified. The identification of
modified
vectors may involve transfer of the DNA of selected transformants to a
nitrocellulose
filter or other membrane and the "lifts" hybridized with kinased synthetic
primer at a
temperature that permits hybridization of an exact match to the modified
sequence but
prevents hybridization with the original strand. Transformants that contain
DNA that
hybridizes with the probe are then cultured and serve as a reservoir of the
modified
DNA.




WO 92/03556 2 0 8 ~ ~ 9 ~ PGT/US91/05753
19
In the constructions set forth below, correct ligations for plasmid
construction
are confirmed by first transforming ~. ~ strain DG101 or another suitable host
with
the ligation mixture. Successful transformants are selected by ampicillin,
tetracycline or
other antibiotic resistance or sensitivity or by using other markers,
depending on the
mode of plasmid construction, as is understood in the art. Plasmids from the
transformants are then prepared according to the method of Clewell gl g_l.,
1969, Pr~c.
N~1. Acid. ~. T~SA x:1159, optionally following chloramphenicol amplification
(Clewell, 1972, ~. Bacte~. ~Q:667). Another method for obtaining plasmid DNA
is
described as the "Base-Acid" extraction method at page 11 of the Bethesda
Research
Laboratories publication Focus, volume 5, number 2, and very pure plasmid DNA
can
be obtained by replacing steps 12 through 17 of the protocol with
CsCI/ethidium
bromide ultracentrifugation of the DNA. The isolated DNA is analyzed by
restriction
enzyme digestion and/or sequenced by the dideoxy method of Singer gl ~., 1977,
Pr~c . 1~1. cad. ~. USA 2:5463, as further described by Messing gl ~., 1981,
T~. Acids ~. Q:309, or by the method of Maxim gl ~., 1980, Methods in
En~,vmologv øx:499.
The control sequences, expression vectors, and transformation methods are
dependent on the type of host cell used to express the gene. Generally,
procaryotic,
yeast, insect, or mammalian cells are used as hosts. Procaryotic hosts are in
general the
most efficient and convenient for the production o~ recombinant proteins and
are
therefore preferred for the expression of Tma polymerasc.
The procaryote most frequently used to express recombinant proteins is ~. ~.
For cloning and sequencing, and for expression of constructions under control
of most
bacterial promoters, E_. Eli K12 strain MM294, obtained from the ~. ~i Generic
Stock Center under GCSC #6135, can be used as the host. For expression vectors
with the PI,N~S control sequence, _E. ~ K12 strain MC1000 lambda lysogen,
N~N53cIg5~ SusPgo, ATCC 3y531, may be used. ~. ~ DG116, which was
deposited with the ATCC (ATCC 53606) on April 7, 1987, and ~. ~ KB2, which
was deposited with the ATCC (ATCC 53075) on March 29, 1985, are also useful
host
cells. For M13 phage recombinants, E_. Eli strains susceptible to phage
infection, such
as _E. Eli K12 strain DG98, are employed. The DG98 strain was deposited with
the
ATCC (ATCC 39768) on July 13, 1984.
However, microbial strains other than ~. ~ can also be used, such as bacilli,
for example Bacilli ~g~,j,~, various species of Pseudomonas, and other
bacterial
strains, for recombinant expression of ~ DNA polymerise. In such procaryotic
systems, plasmid vectors that contain replication sites and control sequences
derived
from the host or a species compatible with the host are typically used
For example, ~. Eli is typically transformed using derivatives of pBR322,
described by Bolivar gJ gl., 1977, Gene 2:95. Plasmid pBR322 contains genes
for




WO 92/03556 PGT/US91/05753
2~894~~
ampicillin and tetracycline resistance. These drug resistance markers can be
either
retained or destroyed in constructing the desired vector and so help to detect
the
presence of a desired recombinant. Commonly used procaryotic control
sequences,
i.e., a promoter for transcription initiation, optionally with an operator,
along with a
5 ribosome binding site sequence, include the ~i-lactamase (penicillinase) and
lactose (lac)
promoter systems (Chang et al., 1977, Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel gl ~1., 1980, Nuc. ci Rg,F. $:4057), and the lambda-
derived PL promoter (Shimatake ~ g~., 1981, Nature ?2:128) and N-gene ribosome
binding site (NHS). A portable control system cassette is set forth in United
States
10 Patent No. 4,711,845, issued December 8, 1987. This cassette comprises a PL
promoter operably linked to the NHS in turn positioned upstream of a third DNA
sequence having at least one restriction site that permits cleavage within six
by 3' of the
NHS sequence. Also useful is the phosphatase A (phoA) system described by
Chang
gl ~1. in European Patent Publication No. 196,864, published October 8, 1986.
15 However, any available promoter system compatible with procaryotes can be
used to
construct a Tma expression vector of the invention.
The nucleotide sequence of the ~ insert may negatively affect the efficiency
of the upstream ribosomal binding site, resulting in low levels of translated
polymerase.
The translation of the Tma gene can be enhanced by the construction of
"translationally
20 coupled" derivatives of the expression vectors. An expression vector can be
constructed with a secondary translation initiation signal and short coding
sequence just
upstream of the ~,a gene coding sequence such that the stop codon for the
short
coding sequence is "coupled" with the ATG start codon for the T-ma gene coding
sequence. A secondary translation initiation signal that efficiently initiates
translation
can be inserted upstream of the Tma gene start codon. Translation of the short
coding
sequence brings the ribosome into close proximity with the Tma gene
translation
initiation site, thereby enhancing translation of the Tma gene. For example,
one
expression system can utilize the translation initiation signal and first ten
colons of the
T7 bacteriophage major capsid protein (gene 10) fused in-frame to the last six
colons
of TrpE. The TGA (stop) colon for TrpE is "coupled" with the ATG (start) colon
for
the Tma gene coding sequence, forniing the sequence TGATG. A one base frame-
shift
is required between translation of the short coding sequence and translation
of the Tma
coding sequence. These derivative expression vectors can be constructed by
recombinant DNA methods.
The redundancy of the genetic code can also be related to a low translation
efficiency. Typically, when multiple colons coding the same amino acid occur,
one of
the possible colons is preferentially used in an organism. Frequently, an
organism
accumulates the tRNA species corresponding to the preferred colons at a higher
level
than those corresponding to rarely used colons. If the pattern of colon usage
differs




WO 92/03556 ~ ~ ~ ~ ~ ~ ~ PGT/U891/05753
21
between Thermotoga maritinna and the host cell, the iRNA species necessary for
translation of the polymerase gene may be in low abundance. In the ~ coding
sequence, arginine is most frequently coded for by the "AG~'~ c~don, whereas
this
codon is used at low frequency in ~. ~ genes, and the corr~.~onding tRNA is
present
in low concentration in ~. ~ host cells. Consequently, the low concentration
in the
~. Eli host cell of "Arg U" tRNA for the "AGA" condon may limit the
translation
efficiency of the polymerase gene RNA in ~. ~i host cells. The efficiency of
translation of the coding sequence within an ~. ~ host cell may be improved by
increasing the concentration of this Arg tRNA species by expressing multiple
copies of
this tRNA gene in the host cell.
In addition to bacteria, eucaryotic microbes, such as yeast, can also be used
as
recombinant host cells. Laboratory strains of Saccharomyces cerevisiae,
Baker's yeast,
arc most often used, although a number of other strains are commonly
available. While
vectors employing the two micron origin of replication are common (Broach,
1983,
Meth. ~,, x:307), other plasmid vectors suitable for yeast expression are
known
(see, for example, Stinchcomb gl ~., 1979, Nature ~$,~:39; Tschempe ~ ~.,
1980,
Gene 1Q:15; . :,nd Clarke ~ ~., 1983, ~. ~. IQ,~:300). Control sequences for
yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess
gl g~.,
1968, ,~. ~y. Enz3rme $gg. 1:149; Holland ~ ~., 1978, ~iotechnolosv x:4900;
and
Holland gl ~., 1981, ~. 4iol. Chem. ~5 :1385). Additional promoters known in
the
art include the promoter for 3-phosphoglycerate kinase (Hitzeman ~ ~., 1980,
~. 'R~1.
~. x:2073) and those for other glycolytic enzymes, such as glyceraldehyde 3-
phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phc~Phoglucose isomerase, and glucokinase. Other
promoters that have the additional advantage of transcription controlled by
growth
wditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome
C,
acid phosphatase, degradative enzymes associated with nitrogen metabolism, and
enzymes responsible for maltose and galactose utilization (Holland, ~).
Terminator sequences may also be used to enhance expression when placed at
the 3' end of the coding sequence. Such terminators are found in the 3'
untranslated
region following the coding sequences in yeast-derived genes. Any vector
containing a
yeast-compatible promoter, origin of replication, and other control sequences
is suitable
for use in constructing yeast r~ expression vectors.
The ~ gene can also be expressed in eucaryotic host cell cultures derived
from multicellular organisms. See, for example, Tissue 1 r , Academic Press,
Cruz
and Patterson, editors (1973). Useful host cell lines include COS-7, COS-A2,
CV-1,
marine cells such as rnurine myelomas NS1 and VERO, HeLa cells, and Chinese
hamster ovary (CHO) cells. Expression vectors for such cells ordinarily
include




WO 92/03556 PCT/US91/05753
~~~~4~5
22
promoters and control sequences compatible with mammalian cells such as, for
example, the commonly used early and late promoters from Simian Virus 40 (S V
40)
(Fiers ~ ~1., 1978, Nature 2:113), or other viral promoters such as those
derived
from polyoma, adenovirus 2, bovine papilloma virus (BPV), or avian sarcoma
viruses,
or immunoglobulin promoters and heat shock promoters. A system for expressing
DNA in mammalian systems using a BPV vector system is disclosed in U.S. Patent
No. 4,419,446. A modification of this system is described in U.S. Patent No.
4,601,978. General aspects of mammalian cell host system transformations have
been
described by Axel, U.S. Patent No. 4,399,216. "Enhancer" regions are also
important
in optimizing expression; these are, generally, sequences found upstream of
the
promoter region. Origins of replication may be obtained, if needed, from viral
sources.
However, integration into the chromosome is a common mechanism for DNA
replication in eucaryotes.
Plant cells can also be used as hosts, and control sequences compatible with
plant cells, such as the nopaline synthase promoter and polyadenylation signal
sequences (Depicker gl g~,., 1982, ~. IVY. $p,~. ~. x:561) are available.
Expression
systems employing insect cells utilizing the control systems provided by
baculovirus
vectors have also been described (Miller el ~1., 1986, Genetic En 'n~ Bering
(Setlow et
~1_., eds., Plenum Publishing) $:277-297). Insect cell-based expression can be
accomplished in $podont~r f ' ei . These systems can also be used to produce
recombinant Tma polymerise.
Depending on the host cell used, transformation is done using standard
techniques appropriate to such cells. The calcium treatment employing calcium
chloride, as described by Cohen, 1972, ~. Natl. Acid. ,5~. ~ x:2110 is used
for procaryotes or other cells that contain substantial cell wall barriers.
Infection with
Aerobacterium tumefaciens (Shaw gl g_l., 1983, ~en~ 2:315) is used for certain
plant
cells. For mammalian cells, the calcium phosphate precipitation method of
Graham and
van der Eb, 1978, Virology ,5:546 is preferred. Transformations into yeast are
carried
out according to the method of Van Solingen ~ ~., 1977, ~. ,~~. ~Q:946 and
Hsiao
g~ g_l., 1979, Proc. Natl. Acid. Sci. ~ 76:3829.
Once the Tma DNA polymerise has been expressed in a recombinant host cell,
purification of the protein may be desired. Although a variety of purification
procedures can be used to purify the recombinant thermostable polymerise of
the
invention, fewer steps may be necessary to yield an enzyme preparation of
equal purity.
Because ~. ~ host proteins are heat-sensitive, the recombinant thermostable
DNA polymerise can be substantially enriched by heat inactivating the crude
lysate.
This step is done in the presence of a sufficient amount of salt (typically
0.3 M
ammonium sulfate) to ensure dissociation of the Tma DNA polymerise from the
host
DNA and to reduce ionic interactions of Tma DNA polymerise with other cell
lysate




20 89495 _
W~I'a/03556 PGT/US91 /05753
23
proteins. In addition, the presence of 0.3 M ammonium sulfate promotes
hydrophobic
interaction with a phenyl sepharose column. Hydrophobic interaction
chromatography
is a separation technique in which substances are separated on the basis of
differing
strengths of hydrophobic interaction with an uncharged bed material containing
hydrophobic groups. Typically, the column is first equilibrated under
conditions
favorable to hydrophobic binding, such as high ionic strength. A descending
salt
gradient may then be used to elute the sample.
According to the invention, an aqueous mixture (containing either native or
necornbinant ~ DNA polymerise) is loaded onto a coluam containing a relatively
strong hydrophobic gel such as phenyl sepharose (manufactured by Pharmacia) or
Phenyl TSK ((manufactured by Toyo Soda). To promote hydrophobic interaction
with
a phenyl sepharose column, a solvent is used that contains, for example,
greater than or
equal to 0.3 M ammonium sulfate, with 0.3 M being preferred, or greater than
or equal
to 0.5 M NaCI. The column and the sample-are adjusted to 0.3 M ammonium
sulfate in
50 mM Tris (pH 7.5) and 1.0 mM EDTA ("TE") buffer that also contains 0.5 mM
DTT, and the sample is applied to the column. The column is washed with the
0.3 M
ammonium sulfate buffer. The enzyme may then be eluted with solvents that
attenuate
hydrophobic interactions, such as decreasing salt gradients, ethylene or
propylene
glycol, or urea. For native ~ DNA polymerise, a preferred embodiment involves
washing the column with 2 M urea and 20% ethylene glycol in TE-DTT.
For long-term stability, r~ DNA polymerise enzyme can be stored in a buffer
that contains one or more non-ionic polymeric detergents. Such detergents are
generally those that have a molecular weight in the range of approximately 100
to
250,000 daltons, preferably about 4,000 to 200,000 daltons, and stabilize the
enzyme
at a pH of from about 3.5 to about 9.5, preferably from about 4 to 8.5.
Examples of
such detergents include those specified on pages 295-298 of McCutcheon's
& Deterrents, North American edition (1983), published by the McCutch~ott
Division --- -
of MC Publishing Co., 175 Rock Road, Glen Rock, NJ (USA) and U.S. Patents
5,352,600 and 5,079,352. -
Preferably, the detergents are selected from the group comprising ethoxylated
fatty alcohol ethers and lauryl ethers, ethoxylated alkyl phenols,
octylphenoxy
polyethoxy ethanol compounds, modified oxyethylated and/or oxypropylated
straight-
chain alcohols, polyethylene glycol monooleate compounds, polysorbate
compounds,
and phenolic fatty alcohol ethers. More particularly preferred are Tween 20, a
polyoxyethylatcd (20) sorbitan monolaurate from ICI Americas Inc., Wilmington,
DE,
and Iconol NP-40, an ethoxylated alkyl phenol (nonyl) from BASF Wyandotte
Corp.
Parsippany, N1.
The thermostable enzyme of ~~is invention may be used for any purpose in
which such enzyme activity is necessary or desired. In a pardcuZarly preferred
B




WO X03556 ~ ~ 8 9 4 9 5 PCT/US91/05753
24
embodiment, the enzyme catalyzes the nucleic acid amplification reaction
lrnown as
PCR. This process for amplifying nucleic acid sequences is disclosed and
claimed in
U.S. Patent Nos. 4,683,202 and 4,865,188. The PCR nucleic acid amplification
method
involves amplifying at least one specific nucleic acid sequence contained in a
nucleic acid
or a mixture of nucleic acids and in the most common embodiment, produces
double-
stranded DNA.
For ease of discussion, the protocol set forth below assumes that the specific
sequence to be amplified is contained in a double-stranded nuclcic-acid.
However, the
process is equally useful in amplifying single-stranded nucleic acid, such as
mRNA,
although in the preferred embodiment the ultimate product is still double-
stranded
DNA. In the amplification of a single-stranded nucleic acid, the first step
involves the
synthesis of a complementary strand (one of the two amplification primers can
be used
for this purpose), and the succeeding steps proceed as in the double-stranded
amplification process described below.
This amplification process comprises the steps of:
(a) contacting each nucleic acid strand with four different nucleoside
triphosphates and two oligonucleotide primers for each specific sequence being
amplified, wherein each primer is selected to be subs:antially complementary
to the
different strands of the specific sequence, such that the extension product
synthesized
from one primer, when separated from its complement, can serve as a template
for
synthesis of the extension product of the other primer, said contacting being
at a
temperature that allows hybridization of each primer to a complementary
nucleic acid
strand;
(b) contacting each nucleic acid strand, at the same time as or after step
(a), with
2S a DNA polymerase from Thermoto~a maritima that enables combination of the
nucleoside triphosphates to form primer extension products complementary to
each
strand of the specific nucleic acid sequence;
(c) maintaining the mixture from step (b) at an effective temperature for an
effective time to promote the activity of the enzyme and to synthesize, for
each different
sequence being amplified, an extension product of each primer that is
complementary to
each nucleic acid strand template, but not so high as to separate each
extension product
from the complementary strand template;
(d) heating the mixture from step (c) for an effective time and at an
effective
temperature to separate the primer extension products from the templates on
which they
3~ were synthesized to produce single-stranded molecules but not so high as to
denature
irreversibly the enzyme;
(e) coolin ~ the mixture from step (d) for an effective time and to an
effective
temperature to promote hybridization of a primer to each of the single-
stranded
molecules produced in step (d); and
B




WO 92/03556 ~ ~ ~ ~ ~ 9 ~ PCT/US91/05753
(f) maintaining the mixture from step (e) at an effective temperature for an
effectivc time to promote the activity of the enzyme and to synthesize, for
each different
sequence being amplified, an extension product of each primer that is
complementary to
each nucleic acid template produced in step (d) but not so high as to separate
each
5 extension product from the complementary strand template. The effective
times and
temperatures in steps (e) and (fj may coincide, so that steps (e) and (f) can
be canned
out simultaneously. Steps (d)-(fj are repeated until the desired level of
amplification is
obtained.
The amplification method is useful not only for producing large amounts of a
10 specific nucleic acid sequence of known sequence but also for producing
nucleic acid
sequences that are known to exist but are not completely specified. One need
know
only a sufficient number of bases at both ends of the sequence in sufficient
detail so that
two oligonucleotide primers can be prepared that will hybridize to different
strands of
the desired sequence at relative positions along the sequence such that an
extension
15 product synthesized from one primer, when separated from the template
(complement),
can serve as a template for extension of the other primer into a nucleic acid
sequence of
defined length. The greater the knowledge about the bases at both ends of the
sequence, the greater can be the specificity of the primers for the target
nucleic acid
sequence and the efficiency of the process.
20 In any case, an initial copy of the sequence to be amplified must be
available,
although the sequence need not be pure or a discrete molecule. In gencral, the
amplification process involves a chain reaction for producing, in exponential
quantities
relative to the number of reaction steps involved, at least one specific
nucleic acid
sequence given that (a) the ends of the required sequence are known in
sufficient detail
25 that oligonucleotides can be synthesized that will hybridize to them and
(b) that a small
amount of the sequence is available to initiate the chain reaction. The
product of the
chain reaction will be a discrete nucleic acid duplex with termini
corresponding to the 5'
ends of the specific primers employed.
Any nucleic acid sequence, in purified or nonpurifted form, can be utilized as
the starting nucleic acid(s), provided it contains or is suspected to contain
the specific
nucleic acid sequence one desires to amplify. The nucleic acid to be amplified
can be
obtained from any source, for example, from plasmids such as pBR322, from
cloned
DNA or RNA, or from natural DNA or RNA from any source, including bacteria,
yeast, viruses, organelles, and higher organisms such as plants and animals.
DNA or
RNA may be extracted from blood, tissue material such as chorionic villi, or
amniotic
cells by a variety of techniques. See, e.g., Maniatis ~ ~., supra, pp. 280-
281. Thus,
the process may employ, for example, DNA or R~IA, including messenger RNA,
which DNA or RNA may be single-stranded or double-stranded. In addition, a DNA-

RNA hybrid that contains one strand of each may be utilized. A mixture of any
of these




WO 92/03556 PGT/US91/05753
26
nucleic acids can also be employed as can nucleic acids produced from a
previous
amplification reaction (using the same or different primers). The specific
nucleic acid
sequence to be amplified can be only a fraction of a large molecule or can be
present
initially as a discrete molecule, so that the spec sequence constitutes the
entire
nucleic acid
The sequence to be amplified need not be present initially in a pure form; the
sequence can be a minor fraction of a complex mixture, such as a portion of
the ~i-
globin gene contained in whole human DNA (as exemplified in Saild gl g~.,
1985,
i n ~Q:1530-1534) or a portion of a nucleic acid sequence due to a particular
microorganism, which organism might constitute only a very minor fraction of a
particular biological sample. The cells can be directly used in the
amplification process
after suspension in hypotonic buffer and heat treatment at about 90 C-100 C
until cell
lysis and dispersion of intracellular components occur (generally 1 to 15
minutes).
After the heating step, the amplification reagents may be added directly to
the lysed
cells. The starting nucleic acid sequence can contain more than one desired
specific
nucleic acid sequence. The amplification process is useful not only for
producing large
amounts of one specific nucleic acid sequence but also for amplifying
simultaneously
more than one different specific nucleic acid sequence located on the same or
different
nucleic acid molecules.
Primers play a key role in the PCR process. The word "primer" as used in
describing the amplification process can refer to more than one primer,
particularly in
the case where there is some ambiguity in the information regarding the
terminal
sequences) of the fragment to be amplified or where one employs the degenerate
primer process of the invention. For instance, in the case where a nucleic
acid
sequence is inferred from protein sequence information, a collection of
primers
containing sequences representing all possible codon variations based on
degeneracy of
the genetic code will be used for each strand. One primer from this collection
will be
sufficiently homologous with the end of the desired sequence to be amplified
to be
useful for amplification.
In addition, more than one specific nucleic acid sequence can be amplified
from
the first nucleic acid or mixture of nucleic acids, so long as the appropriate
number of
different oligonucleotide primers are utilized. For example, if two different
specific
nucleic acid sequences are to be produced, four primers are utilized. Two of
the
primers are specific for one of the specific nucleic acid sequences, and the
other two
primers are specific for the second specific nucleic acid sequence. In this
manner, each
of the two different specific sequences can be produced exponentially by the
present
process.
A sequence within a given sequence can be amplified after a given number of
amplification cycles to obtain greater specificity in the reaction by adding,
after at least




WO 92/03556 ~ ~ ~ 4~ j~ (.~ ~ PCT/US91/05753
27
one cycle of amplification, a set of primers that are complementary to
internal sequences
(i.e., sequences that are not on the ends) of the sequence to be amplified.
Such primers
can be added at any stage and will provide a shorter amplified fragment.
Alternatively,
a longer fragment can be prepared by using primers with non-complementary ends
but
having some overlap with the primers previously utilized in the amplification.
Primers also play a key role when the amplification process is used for ~ vi
mutagenesis. The product of an amplification reaction where the primers
employed are
not exactly complementary to the original template will contain the sequence
of the
primer rather than the template, so introducing an '~~n vitro mutation. In
further cycles,
this mutation will be amplified with an undiminished efficiency because no
further
mispaired priming is required. The process of making an altered DNA sequence
as
described above could be repeated on the altered DNA using different primers
to induce
further sequence changes. In this way, a series of mutated sequences can
gradually be
produced wherein each new addition to the series differs from the last in a
minor way,
but from the original DNA source sequence in an increasingly major way.
Because the primer can contain as part of its sequence a non-complementary
sequence, provided that a sufficient amount of the primer contains a sequence
that is
complementary to the strand to be amplifieti, many other advantages can be
realized.
For example, a nucleotide sequence that is not complementary to the template
sequence
(such as, e.g., a promoter, linker, coding sequence, etc.) may be attached at
the 5' end
of one or both of the primers and so appended to the product of the
amplification
process. After the extension primer is added, sufficient cycles are run to
achieve the
desired amount of new template containing the non-complementary nucleotide
insert.
This allows production of large quantities of the combined fragments in a
relatively
short period of time (e.g., two hours or less) using a simple technique.
Oligonucleotide primers can be prepared using any suitable method, such as,
for example, the phosphotriester and phosphodiester methods described above,
or
automated embodiments thereof. In one such automated embodiment,
diethylphosphoramidites are used as starting materials and can be synthesized
as
described by Beaucage gt ~1-., 1981, Tetrahedron Letters 22:1859-1862. One
method
for synthesizing oligonucleotides on a modified solid support is described in
U.S.
Patent No. 4,458,066. One can also use :: primer that has been isolated from a
biological source (such as a restriction endonuclease digest).




WO 92/03556 PCT/U891/05753
~(~~9~9~
28
No matter what primers are used, however, the reaction mixture must contain a
template for PCR to occur, because the specific nucleic acid sequence is
produced by
using a nucleic acid containing that sequence as a template. The first step
involves
contacting each nucleic acid strand with four different nucleoside
triphosphates and two
oligonucleotide primers for each specific nucleic acid sequence being
amplified or
detected. If the nucleic acids to be amplified or detected are DNA, then the
nucleoside
triphosphates are usually dATP, dCTP, dGTP, and dTTP, although various
nucleotide
derivatives can also be used in the process. The concentration of nucleoside
triphosphates can vary widely. Typically, the concentration is 50 to 200 EtM
in each
dNTP in the buffer for amplification, and MgCl2 is present in the buffer in an
amount
of 1 to 3 mM to activate the polymerise and increase the specificity of the
reaction.
However, dNTP concentrations of 1 to 20 p.M may be preferred for some
applications,
such as DNA sequencing or generating radiolabeled probes at high specific
activity.
The nucleic acid strands of the target nucleic acid serve as templates for the
synthesis of additional nucleic acid strands, which are extension products of
the
primers. This synthesis can be performed using any suitable method, but
generally
occurs in a buffered aqueous solution, preferably at a pH of 7 to 9, most
preferably
about 8. To facilitate synthesis, a molar excess of the two oligonucleotide
primers is
added to the buffer containing the template strands. As a practical matter,
the amount of
primer added will generally be in molar excess over the amount of
complementary
strand (template) when the sequence to be amplified is contained in a mixture
of
complicated long-chain nucleic acid strands. A large molar excess is preferred
to
improve the efficiency of the process. Accordingly, primeraemplate ratios of
at least
1000:1 or higher are generally employed for cloned DNA templates, and primer:
template ratios of about 108:1 or higher are generally employed for
amplification from
complex genomic samples.
The mixture of template, primers, and nucleoside triphosphates is then treated
according to whether the nucleic acids being amplified or detected are double-
or single-
stranded. If the nucleic acids are single-stranded, then no denaturation step
need be
employed prior to the first extension cycle, and the reaction mixture is held
at a
temperature that promotes hybridization of the primer to its complementary
target
(template) sequence. Such temperature is generally from about 35°C to
65°C or more,
preferably about 37°C to 60°C for an effective time, generally
from a few seconds to
five minutes, preferably from 30 seconds to one minute. A hybridization
temperature
of 35°C to 70°C may be used for ~ DNA polymerise. Primers that
are 15
nucleotides or longer in length are used to increase the specificity of primer
hybridization. Shorter primers require lower hybridization temperatures.
The complement to the original single-stranded nucleic acids can be
synthesized
by adding Tma DNA polymerise in the presence of the appropriate buffer, dNTPs,
and




wo nio35s6 2 0 $ ~ ,~ ~ Pcrius9vos~s3
.r.
29
one or more oligonucleotide primers. If an appropriate single primer is added,
the
primer extension product will be complementary to the single-stranded nucleic
acid and
will be hybridized with the nucleic acid strand in a duplex of strands of
equal or
unequal length (depending on where the primer hybridizes to the template),
which may
then be separated into single strands as described above to produce two
single,
separated, complementary strands. A second primer would then be added so that
subsequent cycles of primer extension would occur using both the original
single-
stranded nucleic acid and the extension product of the first primer as
templates.
Alternatively, two or more appropriate primers (one of which will prime
synthesis
using the extension product of the other primer as a template) can be added to
the single-
stranded nucleic acid and the reaction carried out.
If the nucleic acid contains two strands, as in the case of amplification of a
double-stranded target or second-cycle amplification of a single-stranded
target, the
strands of nucleic acid must be separated before the primers are hybridized.
This strand
separation can be accomplished by any suitable denaturing method, including
physical,
chemical or enzymatic means. One preferred physical method of separating the
strands
of the nucleic acid involves heating the nucleic acid until complete (>99%)
denaturation
occurs. Typical heat denaturation involves temperatures ranging from about
80°C to
105°C for times generally ranging from about a few seconds to minutes,
depending on
the composition and size of the nucleic acid. Preferably, the effective
denaturing
temperature is 90°C-100°C for a few seconds to 1 minute. Strand
separation may also
be induced by an enzyme from the class of enzymes known as helicases or the
enzyme
RecA, which has helicase activity and in the presence of riboATP is known to
denature
DNA. The reaction conditions suitable for separating the strands of nucleic
acids with
helicases are described by Kuhn Hoffmann-Berling, 1978, CSH-Quantitative
Biology
x:63, and techniques for using RecA are reviewed in Radding, 1982, ~. $~.
Genetics x:405-437. The denaturation produces two separated complementary
strands
of equal or unequal length.
If the double-stranded nucleic acid is denatured by heat, the reaction mixture
is
allowed to cool to a temperature that promotes hybridization of each primer to
the
complementary target (template) sequence. This temperature is usually from
about
35°C to 65°C or more, depending on reagents, preferably
37°C to 60°C. The
hybridization temperature is maintained for an effective time, generally a few
seconds to
minutes, and preferably 10 seconds to 1 minute. In practical terms, the
temperature is
simply lowered from about 95°C to as low as 37°C, and
hybridization occurs at a
temperature within this range.
Whether the nucleic acid is single- or double-stranded, the DNA polymerase
from ermotog~, maritima can be added prior to or during the denaturation step
or
when the temperature is being reduced to or is in the range for promoting
hybridization.




WO 92/03556 PGT/US91/05753
~U~'~~~~ 30
Although the thermostability of Tma polymerase allows one to add Tim
polymerase to
the reaction mixture at any time, one can substantially inhibit non-specific
amplification
by adding the polymerise to the reaction mixture at a point in time when the
mixture
will not be cooled below the stringent hybridization temperature. After
hybridization,
the reaction mixture is then heated to or maintained at a temperature at which
the activity
of the enzyme is promoted or optimized, i.e., a temperature sufficient to
increase the
activity of the enzyme in facilitating synthesis of the primer extension
products from the
hybridized primer and template. The temperature must actually be sufficient to
synthesize an extension product of each primer that is complementary to each
nucleic
acid template, but must not be so high as to denature each extension product
from its
complementary template (i.e., the temperature is generally less than about
80'C to
90' C).
Depending on the nucleic acids) employed, the typical temperature effective
for
this synthesis reaction generally ranges from about 40°C to
80°C, preferably 50°C to
75'C. The temperature more preferably ranges from about 65'C to 75'C for
'f'hermotog~ maritima DNA polymerise. The period of time required for this
synthesis
may range from about 10 seconds to several minutes or more, depending mainly
on the
temperature, the length of the nucleic acid, the enzyme, and the complexity of
the
nucleic acid mixture. The extension time is usually about 30 seconds to a few
minutes.
If the nucleic acid is longer, a longer time period is generally required for
complementary strand synthesis.
The newly synthesized strand and the complement nucleic acid strand form a
double-stranded molecule that is used in the succeeding steps of the
amplification
process. In the next step, the strands of the double-stranded molecule are
separated by
heat denaturation at a temperature and for a time effective to denature the
molecule, but
not at a temperature and for a period so long that the thermostable enzyme is
completely
and irreversibly denatured or inactivated. After this denaturation of
template, the
temperature is decreased to a level that promotes hybridization of the primer
to the
complementary single-stranded molecule (template) produced from the previous
step,
as described above.
After this hybridization step, or concurrently with the hybridization step,
the
temperature is adjusted to a temperature that is effective to promote the
activity of the
thermostable enzyme to enable synthesis of a primer extension product using as
a
template both the newly synthesized and the original strands. The temperature
again
must not be so high as to separate (denature) the extension product from its
template, as
described above. Hybridization may occur during this step, so that the
previous step of
cooling after denaturation is not required. In such a case, using simultaneous
steps, the
preferred temperature range is 50°C to 70°C.


CA 02089495 2006-10-18
31
The heating and cooling steps involved in one cycle of strand separation,
hybridization, and extension product synthesis can be repeated as many times
as needed
to produce the desired quantity of the specific nucleic acid sequence. The
only
limitation is the amount of the primers, thermostable enzyme, and nucleoside
triphosphates present. Usually, from 15 to 30 cycles are completed. For
diagnostic
detection of amplified DNA, the number of cycles will depend on the nature of
the
sample and the initial target concentration in the sample. For example, fewer
cycles
will be required if the sample being amplified is pure. If the sample is a
complex
mixture of nucleic acids, more cycles will be required to amplify the signal
sufficiently
for detection. For general amplification. and detection, the process is
repeated about I S
times. When amplification is used to generate sequences to be detected with
labeled
sequence-specific probes and when human genomic DNA is the target of
amplification,
the process is repeated 15 to 30 times to amplify the sequence sufficiently so
that a
clearly detectable signal is produced, i.e., so that background noise does not
interfere
with detection.
No additional nucleosides, primers, or thermostable enzyme need be added after
the initial addition, provided that no key reagent has been exhausted and that
the
enzyme has not become denatured or irreversibly inactivated, in which case
additional
polymerise or other reagent would have to be added for the reaction to
continue.
Addition of such materials at each step, however, will not adversely affect
the reaction.
After the appropriate number of cycles has been completed to produce the
desired
amount of the specific nucleic acid sequence, the reaction can be halted in
the usual
manner, e.g., by inactivating the enzyme by adding EDTA, phenol, SDS, or CHC13
or
by separating the components of the reaction.
The amplification process can be conducted continuously. In one embodiment
of an automated process, the reaction mixture can be temperature cycled such
that the
temperature is programmed to be controlled at a certain Ievel for a certain
time. One
such instrument for this purpose is the automated machine for handling the
amplification reaction developed and marketed by Perkin-Elmer Cetus
Instruments.
Detailed instructions for carrying out PCR with the instrument are available
upon
purchase of the instrument.
Tma DNA polymerise is very useful in the diverse processes in which
amplification of a nucleic acid sequence by the polymerise chain reaction is
useful. The
amplification method may be utilized to clone a particular nucleic acid
sequence for
insertion into a suitable expression vector, as described in U.S. Patent No.
4,800,159.
The vector may be used to transform an appropriate host organism to produce
the gene
product of the sequence by standard methods of recombinant DNA technology.
Such
cloning may involve direct Iigation into a vector using blunt-end ligation, or
use of
restriction enzymes to cleave at sites contained within the primers. Other
processes




W0~92,/03556 2 0 8 9 4 9 5 PCT/US91/05753
32
suitable for Tma polymerise include those described in U.S. Patent Nos.
4,683,195 and
4,683,202 and European Patent Publication Nos. 229,701; 237,362; and 258,017.
In
addition, the present enzyme is useful in asymmetric PCR (see Gyllensten and
Erlich,
1988, Proc. Natl. Acid. Sci. USA 85:7652-7656); inverse PCR (Ochman et al.,
1988,
Genetics 120:621); and for DNA sequencing (see Innis et al., 1988, Proc. Natl.
Acid.
Sci. USA 85:9436-9440, and McConlogue et al., 1988, Nuc. Acids Res.
16(20):9869).
Tma polymerise is also believed to have reverse transcriptase activity; see
PCT Patent
Publication No. 91/09944, published July 11, 1991.
The reverse transcriptase activity of ~ DNA polymerise permits this enzyme
to be used in methods for transcribing and amplifying RNA. The improvement of
such
methods resides in the use of a single enzyme, whereas previous methods have
required more than one enzyme. - -
The improved methods comprise the steps of: (a) combining an RNA template
with a suitable primer under conditions whereby the primer will anneal to the
corresponding RNA template; and (b) reverse transcribing the RNA template by
incubating the annealed primer-RNA template mixture with Tma DNA polymerise
under conditions sufficient for the DNA polymerise to catalyze the
polymerization of _
deoxyribonucleoside triphosphates to form a DNA sequence complementary to the
sequence of the RNA template.
In another aspect of the above method, the primer that anneals to the RNA
template may also be suitable for amplification by PCR. In PCR, a second
primer that
is complementary to the reverse transcribed cDNA strand provides a site for
initiation of
synthesis of an extension product. As already discussed above, the Tma DNA
polymerise is able to catalyze this extension reaction on a cDNA template.
In the amplification of an RNA molecule by Tma DNA polymerise, the first
extension reaction is reverse transcription, in which a DNA strand is produced
in the
form of an RNA/cDNA hybrid molecule. The second extension reaction, using the
DNA strand as a template, produces a double-stranded DNA molecule. Thus,
synthesis of a complementary DNA strand from an RNA template with ma DNA
polymerise provides the starting material for amplification by PCR.
When Tma DNA polymerise is used for nucleic acid transcription from an RNA
template, the use of buffers that contain Mn2* provide improved stimulation of
~
3~ reverse transcript3se activity compared to previously used, Mg2* containing
reverse
transcription buffers. Consequently, increased cDNA yields also result from
these
methods.
AS SLatP.d above, the product of Rh'A transcription by Tma DNA polymerise is
an RNA/cDNA hybrid molecule. The RNA is then removed by heat denaturation or
B
...




WO 92/03556 PGT/US91/05753
33
any number of other known methods including alkali, heat, or enzyme treatment.
The
remaining cDNA strand then serves as a template for polymerization of a self
complementary strand, thereby providing a double-stranded cDNA molecule
suitable
for ampl~cation or other manipulation. The second strand synthesis requires a
sequence specific primer and ~ DNA polymerise.
Following the synthesis of the second cDNA strand, the resultant double-
stranded cDNA molecule can serve a number of purposes, including DNA
sequencing,
amplification by PCR, or detection of a specific nucleic acid sequence.
Specific
primers useful for amplification of a scgment of the cDNA can be added
subsequent to
the reverse transcription. Also, one can use a first set of primers to
synthesize a
specific cDNA molecule and a second nested s. . of primers to amplify a
desired cDNA
segment. All of these reactions are catalyzed by ~m DNA polymerise.
~ DNA polymerise can also be used to simplify and improve methods for
detection of RNA target molecules in a sample. In these methods, ~m DNA
polymerise catalyzes: (a) reverse transcription; (b) second strand cDNA
synthesis; and,
if desired (c) amplification by PCR In addition to the improvement of only
requiring a
single enzyme, the use of ~ DIVA polymerise in the described methods
eliminates
the previous requirement of two sets of incubation conditions that were
necessary due
to the use of different enzymes for each procedural step. The use of ~ DNA
polymerise provides RNA transcription and amplification of the resulting
complementary DNA with enhanced specificity and with fewer steps than previous
RNA cloning and diagnostic methods. These methods are adaptable for use in
kits for
laboratory or clinical analysis.
The RNA that is transcribed and amplified in the above methods can be derived
from a number of sources. The RNA template can be contained within a nucleic
acid
preparation from any organism, such as a viral or bacterial nucleic acid
preparation.
The preparation can contain cell debris and other components, purified total
RNA, or
purified mRNA. The RNA template can also be a population of heterogeneous RNA
molecules in a sample. Furthermore, the target RNA can be contained in a
biological
sample, and the sample can be a heterogeneous sample in which RNA is but a
small
portion. Examples of such biological samples include blood samples and
biopsied
tissue sin les.
Aluiough the primers used in the reverse transcription step of the above
methods are generally completely complementary to the RNA template, the
primers
need not be completely complementary. As in PCR, not every nucleotide of the
primer
must anneal to the tcmplate for reverse transcription to occur. For example, a
non-
complementary nucleotide sequence can be present at the 5' end of the primer
with the
remainder of the primer sequence being complementary to the RNA.
Alternatively, non-
complementary bases can be interspersed into the primer, provided that the
primer


CA 02089495 2001-02-05
34
sequence has sufficient complementarity with the RNA template for
hybridization to
occur and allow synthesis of a complementary DNA strand.
The following examples are offered by way of illustration only and are by no
means intended to limit the scope of the claimed invention. In these examples,
all
~~ percentages are by weight if for solids and by volume if for liquids,
unless otherwise
noted, and all temperatures are given in degrees Celsius.
EXAMPLE 1
Purification of Thermotoga maritima
DNA Pol,~vr
This example describes the isolation of Tma DNA polymerise from Thermoto~a
maritima. The DNA polymerise was assayed at various points during ptu~ificadon
according to the method described for T~ca polymerise with one modification (
1 mM
MgCl2) in Lawyer~t al., 1989,1. Biol. hem. ~(11):C427-6437.
Typically, this assay is performed in a total volume of 50 ltl of a reaction
mixture composed of 2~ mM TAPS-HCI, pH 9.5 (20'C); 50 mM KCI; 1 mM MgCl2; 1
mM ~i-mercaptoethanol; 200 ~M in each of dATP, dGTP, and TTP; 100 ~M a-32P-
dCTP (0.03 to 0.07 ~Ci/nMol); 12.5 ~tg of activated salmon sperm DNA; and
polymerise. The reaction is initiated by addition of polymerise in diluent
(diluent is
composed of 10 mM Tris-HCI, pH $.0, 50 mM KC1, 0.1 mM EDTA, 1 mg/ml
autoclaved gelatin, 0.5% NP40, 0.5% Tween 20, and 1 mM ~i-mercaptoethanol),
and
the reaction is carried out at 75°C. For the calculations shown below,
one assumes that
the volume of the polymerise (and diluent) added is ~ ~t.l, and the total
reaction volume
is SO ~tl. After a 10 minute incubation, the reaction is stopped by adding 10
E.tl of 60
mM EDTA. The reaction mixture is centrifuged, and 50 ~tl of reaction mixture
is
transferred to 1.0 ml of 50 ~tg/ml carrier DIVA in 2 m.M EDTA (at 0°C).
An equal
volume (1 ml) of 20% TCA, 2% sodium pyrophosphate is added and mined. The
mixture is incubated at 0°C for 15 to 20 minutes and then filtered
through Whitman
GF/C filters and extensively was;~ed (6 x ~ ml) with a cold mixture containing
5% TCA
and 1 % pyrophosphate, followed by a cold 9~% ethanol wash. The filters are
then
dried and the radioactivity counted. Background (minus enzyme) is usually
0.001% to
0.01% of input cpm. About ~0 to 250 pmoles 3~P-dCTP standard is spotted for
unit
calculation. One unit is equal to 10 nmoles dIVTP in;orporated in 30 minutes
at 7~°C.
Units are calculated as follows.
sample cnm - enzyme dil. cpm - pmole dCTP incorporated
'specific activiry of dCTP
(cpm/pmole j




WO 92/03556 PCT/US91 /05753
,..\ ZQ 89495
nmole incorporated x 3 x dilution factor x 4 = units/ml
4.167 x 10
The 4.167 factor results from counting only 5/6 (50 ~tl) of the reaction
volume after the
stop solution is added (60 ul).
5 All operations were carried out at 0'C to 4'C unless otherwise stated. All
glassware was baked prior to use, and solutions used in the purification were
autoclaved, if possible, prior to use.
About 50 g of frozen Thermotoaa maritima strain MSBS cells (provided by
Prof. Dr. K. O. Stetter, Regcnsburg, Germany) were thawed in 25 ml of 3 x TE-
DTT
.,10 buffer (150 mM Tris-Cl, pH 7.5, 3 mM EDTA, and 3 mM dithiothreitol)
containing
2.4 mM PMSF (from 144 mM stock in DMF) and homogenized at low speed with a
magnetic stirrer. The thawed cells were lysed in an Aminco french pressure
cell (8 -
20,000 psi). The lysate was diluted with additional 1 x TE-DTT buffer
containing
fresh 2.4 mM PMSF to final 5.5x cell wet weight and sonicatcd to reduce
viscosity (40
15 to 100% output, 9 min., 50% duty cycle).
The resulting fraction, fraction I (275 ml) contained 5.31 g of protein and
15.5 x 104 units of activity. Ammonium sulfate was added to 0.2 M (7.25 g) and
the
lysate stirred for 15 mic:.rtes on ice. Ammonium sulfate prevents the Tma DNA
polymerise from binding to DNA in the crude lysate and reduces ionic
interactions of
20 the DNA polymerise with other cell lysate proteins.
Empirical testing showed that 0.2% Polymin P (polyethylcneimine, PEI)
precipitates __>9290 of the total nucleic acid. Polymin P (pH 7.5) was added
slowly to
0.2% (5.49 mI of 10% PEI) and the slurry stirred 30 minutes on ice, then
centrifuged at
30,000 x g at 4'C for 30 minutes. The supernatant was designated fraction II
(246 ml)
25 and contained 3.05 g of protein and 12.5 x 104 units of activity.
Fraction II was adjusted to 0.3 M.ammonium sulfate by addition of 3.24 g solid
ammonium sulfate to ensure complete binding of the DNA polymerise to phenyl
sepharos~ Fraction II was then loaded onto a 2.2 x 6.6 cm (25 ml) phenyl
sepharose
CL-4B (lot OM 08012, purchased from Pharmacia - LKB) column (equilibrated in
TE
30 containing 0.3 M ammonium sulfate and 0.5 mM DTT) at 38 ml/hr (10
ml/em2/hr). All
resins were equilibrated and recycled according to the manufacturer's
recommendations.
The column was washed with 150 ml of the same buffer (A~a to baseline),
then with 90 ml TE containing 0.5 mM DTT (no ammonium sulfate), followed by a
35 wash with 95 ml of 20% ethylene glycol in TE containing 0.5 mM DTT and
finally,
eluted with 2 M urea in TE containing 20% ethylene glycol and 0.5 mM DTT. When
the column fractions were assayed, a large proportion of the activity was
found in the
flow-through and wash fractions, indicating that the capacity of the column
had lien
exceeded. Approximately 70% of the DNA polymerise which had bound to this
first
B




WO 03556 2 p g g 4 9 5 PCT/US91/05753
36
phenyl sepharose column eluted at low salt (with the TE-DTT wash), and the
balance of
the bound material eluted with 2 M urea in 20% ethylene glycol in TE-DTT wash.
The flow-through activity from the first phenyl sepharose column was
designated PSII load (226 ml) and contained 1.76 g protein. Fraction PSII load
was
S applied to a second phenyl sepharose column (of the same lot and
dimensions), and the
run was repeated the same way. Again, the capacity of the column was exceeded,
and
activity was found to elute with both the low salt and 2 M urea washes. Only
10% of
the bound DNA polymerise eluted with the TE-DTT wash; the major portion (--
90%)
eluted with the 2 M urea in 20% ethylene glycol in TE-DTT wash.
The flow-through activity from the second phcnyl sepharose column was
combined with the TE-DTT eluates from the first and second phenyl scpharose
columns
and adjusted to 0.3 M ammonium sulfate. This fraction (PSIII load, 259.4 ml)
contained 831 mg protein and was reapplied to a third phenyl sepharose column
of 50
ml bed volume at 10 mUcm2/hr. This time; all of the applied activity was
retained by
the column and only eluted with the 2 M urea in 20% ethylene glycol in TE-DTT
wash.
All three urea eluates werc separately concentrated -3 to 4 fold on Amicon
YM30 membranes and dialyzed into heparin sepharose loading buffer shortly
after
elution to avoid prolonged exposure to urea (to avoid carbamylation). The
dialyzed and
concentrated urea eluates were assayed for protein concentration and wcre
found to
vary greatly in their specific activity. Because the urea eluate from the
second phenyl
sepharose column contained the majority of the activity at significantly
higher specific
activity (--8 x 104 units of activity at -1000 units/mg protein) than the
other two eluates,
it was processed separately from thcm.
The dialyzed and concentrated phenyl sepharose II urea eluate was applied to a
S ml bed volume heparin sepharose CL 6B (purchased from Pharmacia - LKB)
column
that had been equilibrated with 0.08 M KCI, 50 mM Tris-Cl, pH 7.5, 0.1 mM
EDTA,
0.2% Tween 20, and 0.5 mM DTT. This column and all subsequent columns were run
at 1 bed volume per hr. All of the applied DNA polymerise activity was
retained by the
column. The column was washed with 17 ml of the same buffer (AZ8o to baseline)
and
eluted with 60 ml of a linear 80 to 500 mM KCl gradient in the same buffer.
Fractions (0.53 ml) eluting between 0.21 and 0.315 M KCl were analyzed by
SDS-PAGE. The peak fractions eluting between 0.225 and 0.275 M KCl were pooled
separately. The flanking fractions~w~re kept to be combined later with other
fractions.
The pool of peak fractions (affigel'I load) was diluted with affigel-blue
buffer without
KCl to reduce its ionic strength to 0.15 M KC1.
The affigel I load fraction contained 3.4 mg of protein and was applied to a
4.3
ml affigel-blue (purchased from BioRad) column, which had becn equilibrated in
25
mM Tris-Cl, pH 7.5, 0.1 mM EDTA, 0.2% Tween 20, 0,5 mM DTT, and 0.15 M
KCI. All of the applied Tma DNA polymerise was retained. The column was washed
B




W~2/03556 ~ ~ 8 ~ ~ ~ PCT/US9l/Of753
37
with 15 ml of the same buffer and eluted with a 66 ml linear 0.1 S to 0.7 M
KC1
gradient in the same buffer.
Fractions (0.58 ml) eluting between 0.34 and 0.55 M KCl were analyzed by
SDS-PAGE and appeared to be >90% pure. The polymerise peak fractions were no
5 longer contaminated with site-specific endonuclease (indicated by absence of
lower-
molecular-weight specific DNA fragments after one or twenty-two hours
incubation at
65'C with 2 units of Tma polymerise using 600 ng of plasmid pLSG 1 (ccc-DNA)).
The polymerise peak fractions eluting between 0.3 and 0.55 M were pooled and
_ concentrated ~20-fold on an Amicon YM 30 membrane. This fraction was then
diafiltered into 2.5 x storage buffer (50 mM Tris-Cl, pH ?.5, 250 mM KCI, 0.25
mM
EDTA, 2.5 mM DTT, and 0.5% Tween 20 [Pierce, Surfact-Amps]) and stored at 4'C.
The urea eluates from the first and third phenyl sepharose columns were
combined with the flanking fractions from the first heparin sepharose column.
This
pool (HSII load) contained --200 mg protein and wis diluted with heparin
sepharosc
buffer without KCl to adjust its ionic strength to 80 mM KCl. HSII load was
applied .
to a 16 ml bed volume heparin sepharose column (equilibrated in 80 mM KC1, 50
mM'-
Tris-Cl, pH 7.5, 0.1 mM EDTA, 0.2% Tween 20, and 0.5 mM DTT). No detectable
polymerise acrivity appeared in the flow-through fractions.
The column was washed with 80 ml of the same buffer and eluted with a 200
ml linear 80 to 750 mM KCl gradient in the same buffer. Fractions (2 ml)
eluting
between 0.225 and 0.335 M KCl were combined, concentrated --5-fold on an
Amicon
YM 30 membrane, and dialyzed into hydroxyapatice-buffer. This fraction (HA
load)
contained 9.3 mg protein and was loaded onto a 4 ml bed volume hydroxyapatite
(high
resolution HPT, purchased from Calbiochem) column that had been equilibrated
in 10
mM potassium phosphate buffer, pH 7.5, 0.5 mM DTT, 0.1 mM EDTA, and 0.2%
Tween 20. All of the applied DNA polymerise activity was retained by the
column.
The column was washed with 12 ml of the same o;.iffer and eluted with a 60 ml
linear 10 to 500 mM potassium phosphate (pH 7.5) gradient. Fr. ~ ~tions (0.8
ml)
eluting between 0.105 and 0.230 M potassium phosphate were ~ .alyzed by SDS
PAGE. Compared to the affig~l column I load fraction (which by SDS-PAGE
appeared to be ~10 to 20% pure), these fractions were ~5-fold less pure. The
DNA
polymerise peak fractions eluting between 0.105 and 0.255 M potassium
phosphate
were combined, concentr4~ed -3-fold on an Amicon YM 30 membrane, and
diafiltered
into affigel-blue buffer.
The affigel II load fraction was applied to a 3 ml bed volume affigel-blue
column that had been equilibrated in affigel-blue buffer. No detectable DNA
polymerise activity appeared in the flow-through fractions. The column was
washed
with 9 ml of the same buffer and eluted with a 50 ml linear 0.2 to 0.7 M KCl
gradient
in :he same buffer. Fractions (0.58 ml) eluting between 0.33 and 0.505 M KCl
were
B



PGT/US91/05753
-..,
WO 92/03556
20~949~ 38
analyzed by SDS-PAGE. Because the earlier eluting fractions looked slightly
cleaner
by their silver staining pattern, two pools were made. Fractions eluting
between 0.31
and 0.4 M KCl were combined into pool I; fractions eluting between 0.4 and
0.515 M
KCl were combined into pool II. The two pools were each separately
concentrated ~7-
fold on an Amicon YM 30 membrane.
All three affigel-blue pools still contained high levels of contaminating, non-

specific nucleases. Upon incubation at 70°C with 1.5 units of DNA
polymerise, both a
single-strand M13 DNA template and a multifragment restriction digest of a
plasmid
were degraded within a few hours. ~n_ i~-activity gels were run and showed
that the
DNA polymerise fractions had not suffered proteolytic degradation.
The two pools from the second affigel-blue column were combined and
dialyzed into a phosphocellulose column buffer. The dialyzed fraction (Pll I
load) was
loaded onto a 3 ml phosphocellulose column, which had been washed overnight
with
25 mM Tris-Cl, pH 7.5, 50 mM KCI, 0.1 mM EDTA, 0.2°Io Tween 20, and 0.5
mM
DTT. This wash later proved to have been insufficient to equilibrate the pH of
the
phosphocellulose resin. Unfortunately, this was discovered after the sample
had been
loaded onto the column. All of the applied activity bound to the column.
The column was washed with 9 ml of loading buffer and eluted with a 45 ml
linear 50 to 700 mM KCl gradient. DNA polymerise peak fractions (0.58 ml)
eluting
between 0.46 and 0.575 M KCl were analyzed by SDS-PAGE.
Separation of contaminating proteins was observed throughout the peak: a ~-45
kDa contaminating band elutes at 0.53 M KCI; an ~85 kDa contaminating band has
an
elution peak at 0.54 M KCI. Therefore, this column was repeated (loading at
somewhat higher ionic strength considering the elution profile of the
polymerise). The
peak fractions, eluting between 0.475 and 0.56 M KCl from the first
phosphocellulose
column were combined with the pool from the first affigel column. The combined
fraction (Pll II load) now contained all of the purified polymerise (~7.5 x
104 units).
Fraction Pll II load was diluted with phosphocellulose buffer to adjust its
ionic
strength to 0.2 M KCI. Pll Il load was loaded onto a 9 ml bed volume
phosphocellulose column, which, this time, had been equilibrated to the
correct pH and
ionic strength of 25 mM Tris-Cl, pH 7.5, 200 mM KCI, 0.1 mM EDTA, 0.2% Tween
20, and 0.5 mM DTT. The column was washed with 27 ml of the same buffer and
was
intended to be eluted with a 140 ml linear 0.2 to 0.8 M KCl gradient. However,
instead of an upper limit buffer of 0.8 M KCI, the buffer had a concentration
of 52 mM
KCl which resulted in a gradient decreasing in salt. The column was then
reequilibrated with 32 ml of 0.2 M KCl-phosphocellulose buffer, and the 140 ml
linear
0.2 to 0.8 M KCl gradient was reapplied.
The routine assays of flow-through, wash, and gradient fractions showed that,
at this higher pH (pH 7.5), the DNA polymerise does not bind to the
phosphocellulose




WO 92/03556 ~ ~ ~ PGT/US91/05753
39
resin at 0.2 M KCI. The DNA polymerise activity containing fractions from the
flow-
through, wish, and decreasing salt-gradient-fractions were combined. The
resulting
pool was concentrated on an Amicon YM30 membrane. However, a mishap with the
concentrator led to further losses of DNA polymerise activity. The recovered
activity
was dialyzed into phosphocellulose buffer with 50 mM KCl and designated Pll
III load.
This fraction was loaded onto a 5 ml bed volume phosphocellulose column that
had been equilibrated with phosphocellulose buffer with 50 mM KCI. All of the
applied activity was retained by the column. The column was washed with 15 ml
of the
same buffer and eluted with a 45 ml linear 50 to 500 mM KCl gradient in the
same
buffer. Fractions (0.87 ml) eluting between 0.16 and 0.33 M KCl were analyzed
by
SDS-PAGE and ~ ~ activity gels.
Based on the silver staining pattern, two pools were made. The peak fractions,
eluting between 0.215 and 0.31 M KCI, were kept separate from the leading and
trailing fractions, which were combined into a side-fractions pool. Both pools
were
concentrated on centricon 30 membranes and diafiltered into 2.5x storage
buffer (50
mM Tris-HCI, pH 7.5, 250 mM KCI, 0.25 mM EDTA, 2.5 mM DTT, and 0.5%
Tween 20 [Pierce, Surfact-Amps]) and subsequently mixed with 1.5 volumes of
80%
glycerol.
About 3.1 x 104 units were recovered in the peak fraction; the side pool
yields
an additional 1 x 103 units of activity. The purified DNA polymerise was
undegraded
as evidenced by an unchanged migration pattern in an '~ ~ activity gel. The
molecular weight as determined by gel electrophoresis of the purified DNA
polymerise
is approximately 97 kDa. ~ DNA polymerise is recognized by epitope-specific
antibodies that correspond to ,T~a DNA polymerise amino acid residues number
569
through 587 (DGTP1) and 718 through 732 (DGTP3).
Synthetic oligodeoxyribonucleotides DG 164 through DG 167 are four different
16-fold degenerate (each) 22mer pools designed as "forward" primers to one of
the
motifs in the template binding domains (3'-most 14 nucleotides) of
thermostable DNA
polymerises. This motif is the amino acid sequence_Gly-Tyr-Val-Glu-Thr and
corresponds identically to the T. a~uaticus (~) DNA polymerise amino acids 718
through 722 and to the ~. lhermophilus (~ DNA polymerise amino acids 720
through 724. This motif is found in a DNA polymerise gene in all Thermus
species.
The combined primer pool is 64-fold degenerate, and the primers encode a
,B~III
recognition sequence at their 5'-ends.
Forward primers DG 164 through DG 167 are shown below:




p~T/US91/05753
WO 92/03556
DG164 SEQ B7 NO: 2 5'CGAGATCTGGNTAYGTWGAAAC
DG165 SEQ ID NO: 3 5'CGAGATCTGGNTAYGTWGAGAC
DG 166 SEQ ID NO: 4 5'CGAGATCTGGNTAYGTSGAAAC
DG167 SEQ ID NO: 5 5'CGAGATCTGGNTAYGTSGAGAC
5 In these forward primers: A is Adenine; C is Cytidine; G is Guanidine; T is
Thymine;
Y is C+T (pYrimidine); S is G+C (Strong interaction; 3 H-bonds); W is A+T
(Weak
interaction; 2 H-bonds); and N is A+C+G+T (aNy).
Synthetic oligodeoxyribonucleotides DG 160 through DG 163 are four different
8-fold degenerate (each) 20mer pools designed as "reverse" primers to one of
the
10 motifs in the template binding domains (3'-most 14 nucleotides) of
thermostable DNA
polymerises. These primers are designed to complement the (+)-strand DNA
sequence
that encodes the motif Gln-Val-His-Asp-Glu and that corresponds identically to
the Tea
DNA polymerise amino acids 782 through 786 and to the T t DNA polymerise amino
acids 784 through 788. This motif is found in a DNA polymerise gene in all
~'hermus
15 species. The combined primer pool is 32-fold degenerate, and the primers
encode an
SRI recognition sequence at their 5'-ends.
Reverse primers DG 160
through 163 are shown
below:


DG160 SEQ m NO: 6 5'CGGAATTCRTCRTGWACCTG


DG161 SEQ ID NO: 7 5'CGGAATTCRTCRTGWACTTG


20 DG 162 SEQ m NO: 5'CGGAATTCRTCRTGSACCTG
8


DG163 SEQ ID NO: 9 5'CGGAATTCRTCRTGSACTTG


In these reverse primers A, C, G, T, S, and W are as defined above, and R is
G+A
(puRine).
To amplify an ~230bp fragment of the Tma DNA polymerise gene, a PCR
25 amplification tube was prepared without MgCl2 that contained in 80 N.l: (1)
5 ng
denatured Tma genomic DNA; (2) 50 pmoles (total) of the combined forward
primer set
DG164-DG167; (3) 50 pmoles (total) of the combined reverse primer set DG160-
DG163; (4) 2 units ~ DNA polymerise; (5) 50 p.M each (final) dNTP; (6) 0.05%
Laureth-12; and (7) standard PCR buffer except no magnesium chloride.
30 The sample was flash-frozen at -70'C and then stored at -20'C. The frozen
sample was carefully layered with 20 ~t.l of IOmM MgCl2 (final concentration 2
mM),
immediately overlayed with 50 ~tl of mineral oil, and cycled in a Perkin Elmer
Cetus
Thermal Cycler according to the following file: ( 1 ) step to 98°C -
hold 50 seconds; (2)
step to 50'C - hold 10 seconds; (3) ramp to 75'C over 4 minutes; and (4) step
to 98'C.
35 The file was repeated for a total of 30 cycles. One-fifth (20 ~.1) of the
amplification
product was purified on a 3% Nusieve/1% Seakem agarose composite gel, and the
approximately 230 by fragment was eluted, concentrated, and digested with
Bgl_II and
EcoRI.




WO 92/03556 PCT/US91/05753
2~~~~~5~
41
Synthetic oligodeoxyribonucleoddes DG 154 and DG 155 are two different 32-
fold degenerate (each) l9mer pools designed as "forward" primers to one of the
motifs
in the primeraemplate binding domains (3'-most 11 nucleotides) of thermostable
DNA
polymerises. This motif is the tetrapeptide sequence Thr-Ala-Thr-Gly and
corresponds
identically to the ~ DNA polymerise amino acids 569 through 572 and to ~ DNA
polymerise amino acids 571 through 574. This motif is found in a DNA
polymerise
gene in all Thermos species. The combined primer pool is 64-fold degenerate
and the
primers encode a ,~g~II recognition sequence at their 5'-ends.
Forward primers DG 154 and DG 155 are presented below:
DG154 SEQ m NO: 10 CGAGATCT~ CNGCNACWGG
DG155 SEQ ID NO: 11 CGAGATCTACNGCNACSGG
In these forward primers, A, C, G, T, S, W, and N are as defined above.
To amplify an approximately -667bp fragment of the ~ DNA polymerise
gene, a PCR amplification tube was prepared without MgCl2 that contained, in
80 E,tl:
(1) 5 ng denatured ~ genomic DNA; (2) 50 pmoles (total) o~ she combined
forward
primer set DG154-DG155; (3) 50 pmoles (total) of the combined reverse primer
set
DG160-DG163; (4) 2 Units of ~q DNA polymerise; (5) 50 ~,M each (final) dNTP;
(6)
0.05% Laureth 12; and (7) standard PCR buffer except no magnesium chloride.
The sample was flash-frozen at -70°C and then stored at -
20°C. The frozen
sample was carefully layered with 20 ~tl of 10 mM MgCl2 (final concentration 2
mM),
immediately overlayed with 50 ~tl of mineral oil, and cycled in a Perldn Elmer
Cetus
Thermal Cycler according to the following file: (1) step to 98°C - hold
50 seconds; (2)
step to 55°C - hold 10 seconds; (3) ramp to 75°C over 4 minutes;
(4) step to 98°C. The
file was repeated for a total of 30 cycles.
One-fifth 20 ~tl) of the amplification product was purified on a 1.5% agarose
gel, and the approximately 670 by fragment was eluted, concentrated, and
digested
with $g~II and SRI as above.
These amplification reactions yielded a 667 by fragment and a 230 by fragment,
which was a subfragment of the 667 by fragment. These fragments proved useful
in
obtaining the complete coding sequence for the Tma DNA polymerise I gene, as
described in the following example.
EXAMPLE 3
Cloning the Thermotoga maritima fTma)
DNA Polvmerase I Gene
This Example describes the strategy and methodology for cloning the Tma DNA
polymerise I (Tma Pol I) gene of Thermoto~_a maritima.
The DNA sequences of the PCR products generated with primers DG164-167
and DG 160-163 (230 bp) and DG 154, 155 and DG 160-163 (667 bp) contain an
XmaI




WO~/03556 ~ PCT/US91/05753
2089495
42
restriction site recognition sequence, 5'CCCGGG. Oligonucleotides were
designed to
hybridize to sequences upstream and downstream of the Xmal site. DG224 is a
2lmer,
homologous to the PCR products 59-79 by 3'-distal to the Xmal site. DG225 is a
22mer, homologous to the PCR products from the r~I site to 2lbp upstream (5')
of
the Xmal site. The sequence of DG224 and of DG225 is shown below (K is G or
T).
DG224 SEQ ID NO: 12 5'ACAGCAGCKGATATAATAAAG
DG225 SEQ ID NO: 13 5'GCCATGAGCTGTGGTATGTCTC
DG224 and DG225 were labelled by tailing with biotin-dUTP and terminal
transferase
in reactions designed. to add approximately 8 biotin-dUMP residues to the 3'-
end of
oligonucleotides. These labelled oligonucleotides were used as probes in
Southern blot
analysts of restriction digests of genomic ~ DNA. A preliminary restriction
map
was generated based on the Southern analysis results, and the DNA sequences of
the
PCR products that were generated as described in Example 2.
The preliminary map showed that the entire DNA polymerise gene is
contained in two XmaI fragments. Most of the gene, including the 5'-end,
resides on ~
an approximately 2.6 kb ~,I fragment. The remainder of the gene (arid the 3'-
end)
resides on an approximately 4.2 kb ~I fragment. The two ~I fragments
containing the entire Tma DNA polymerise gene were cloned into plasmid pBS
13+"~
(also called pBSMl3+j as described below. ,
About 40 micrograms of ~m genomic DNA were digested to completion with
Xm . The Xmal digest was size-fractionated via electroelution. Slot blot
analyses of
a small portion of each fraction, using Y 32p-ATp-kinased DG224 and DG225
probes,
identified the fractions containing the 4.2 kb 3'-fragment (hybridizing with
DG224) and
the 2.6 kb 5'-fragment (hybridizing with DG225). Fractions were concentrated
via
ethanol precipitation and then ligated with Xmai-digested pBS 13+
(Stratagene).
Ampicillin-resistant transformants were selected on nitrocellulose filters and
the filters
probed with Y-32P-ATP-kinascd DG224 or DG225 probe as appropriate. Plasmid
DNA was isolated from colonies that hybridized with probe. Restriction
analysis was
performed to confirm that fragments were as expected and to dcterinirte
orientation of
fragments relative to the pBS 13+ vector.
DNA sequence analysis of the cloned fragments was performed using the
"universal" and "reverse" sequencing primers (which prime in the vector,
outside the
restriction site polylinker region). In addition, for ~'-clones, the primers
used to
determine the DNA sequence of the DG154-1~5/DG160-163 667 by PCR clone were
employed. Preliminary DNA sequence analysis confirmed that the desired DNA
fragments containing the Tma DNA polymerise gene had been cloned
From the preliminary DNA sequence, further sequencing primers were
designed to obtain DNA sequence of more internal regions of the fragments. In
addition, to facilitate D:~A sequence analysis, several deletions of the two
XmaI
B




WO 92/03556 PCT/US91/05753
20 89495
43
fragments were made. For both orientations of the 2.6 kb 5'-fragment, EcoRI,
SacI,
and X~I digests were each diluted and ligated under conditions that favored
intramolecular ligation, thus deleting DNA between the vector F~gRI, ~I, and
X~I
sites and the corresponding sites in the ~ ~I fragment. Such internal
deletions
allow ready DNA sequence analysis using the "universal" or "reverse"
sequencing
primers.
Similarly, a deletion of the 4.2 kb 3'-fragment was made, fusing the ~~HI
site of the vector with the III site approximately 650 by from the ~ Pol I
internal
~I site in that clone ($~r~r HI and $gIII have identical GATC cohesive ends
that
ligate readily with one another). This deletion allows for DNA sequence
analysis of the
3'-end of the ~ Pol I gene.
Restriction site analysis reveals that both the 2.6 kb 5'-fragment and the 4.2
kb
3'-fragment lack NcoI, l~leI, and ~I restriction sites. Knowing the ATG start
and
coding sequence of the Tma Pol I gene, one can design oligonucleotides that
will alter
the DNA sequence at the ATG start to include an l~gI, T~I, or ~I restriction
site ._
via oligonucleotide site-directed mutagenesis. In addition, the mutagenic
oligonucleotides can be designed such that a deletion of sequences between the
j~
promoter in the pBS 13+ vector and the beginning of the Tma Pol I gene is made
concurrent with the inclusion of an T~eI or t~gI recognition sequence at the
ATG start.
The deletion of sequences between the l~c promoter in the vector and start of
the
r~ Pol I gene would also eliminate the restriction site in the deleted region,
thus
making it convenient to assemble the entire coding sequence in an expression
plasmid
using conventional skill in the art (see, e.g., synthesis protocols for pDG
174 - pDG 181
in U.S. 5,789,224, U.S. 5,693,517, U.S. 5,641,864, U.S. 5,618,711, U.S.
5,561,058,
U,S. 5,407,800 and U.S. 5,310,652 (Patent Family A), and Example 5).
EXAMPLE 4
PCR With Tma DNA Po erase
About 1.25 units of the ~ DNA polymerise purified in Example 1 is used to
amplify rRNA sequences from Tth genomic DNA. The reaction volume is 50 ~tl,
and
the reaction mixture contains 50 pmol of primer DG73, 105 to 106 copies of the
Tth
genome (--2 x 105 copies of genome/ng DNA), 50 pmol of primer DG74, 200 ~Nl of
each dNTP, 2 mM MgCl2, 10 mM Tris-HCI, pH 8.3, 50 mM KCI, and 100 ~tg/ml
gelatin (optionally, gelatin may be omitted).
The reaction is carried out on a Perkin-Elmer Cetus Instruments DNA Thermal
Cycler. Twenty to thirty cycles of 96'C for 15 seconds; 50'C for 30 seconds,
and
75'C for 30 seconds are carried out. At 20 cycles, the amplification product
(160 by in
size) can be faintly seen on an ethidium bromide stained gel, and at 30
cycles, the
product is readily visible (under UV light) on the ethidium bromide stained
gel.
B




WO 92/03556 ~ ~-~, PGT/US91/05753
-.~,.
~~95
.Y
44
The PCR may yield fewer non-specific products if fewer units of T~ DNA
polymerise are used (i.e., 0.31 units/50 wl reaction). Furthermore, the
addition of a
non-ionic detergent, such as laureth-12, to the reaction mixture to a final
concentration
of about 0.5% to 1% can improve the yield of PCR product.
Primers DG73 and DG74 are shown below:
DG73 SEQ 117 NO: 14 5' TACGTTCCCGGGCCTTGTAC
DG74 SEQ ID NO: 15 5' AGGAGGTGATCCAACCGCA
Vectors for Tma DNA Polvmerase
A. Mutasenesis of the 5' and 3' Ends of the Tma Pol I Gene
The 5' end of the Tma gene in vector pBS:Tma7-1 (ATCC No. 68471, later
renamed pTma01) was mutagenized with oligonucleotides DG240 and DG244 via
oligonucleotide site-directed mutagenesis. Plasmid pBS:Tma7-1 consists of the
2.6 kb
5' I fragment cloned into vector pBS 13+. Resultant mutants from both
mutageneses had deletions between the ATG of (3-galactosidase in the pBS+
vector and
the ATG of ~ Pol I so that the Tma coding sequence was positioned for
expression
utilizing the vector ~ promoter, operator, and ribosome binding site (RBS).
Both sets
of mutants also had alterations in the second and sixth colons for Tma Pol I
to be more
compatible with the colon usage of ,~. Eli without changing the amino acid
sequence
of the encoded protein. In addition, DG240 placed an ~I restriction site at
the ATG
start of the coding sequence (5'CATATGI, and DG244 placed an ,~I restriction
site
at the ATG start of the coding sequence (5'CCATGG). DG240 mutant candidate
colonies were screened with [y32P]-labelled oligonucleotide DG241, and DG244
mutant candidate colonies were screened with [y32P]-labelled oligonucleotide
DG245.
Plasmid DNA was isolated from colonies that hybridized with the appropriate
probes,
and mutations were confirmed via restriction analysis and DNA sequence
analysis. The
DG240 mutant was named pTmaS'Nde#3 and later renamed pTma06. The DG244
mutant was named pTmaS'Nco#9 and later renamed pTma07.
The 3'-end of the Tma Pol I gene was mutagenized in pBSTma3'11-1 BamBgl
(ATCC No. 68472, later renamed pTma04) with mutagenic oligonucleotide DG238.
Plasmid pBSTma3'11-1 BamBgl was constructed as described in Example 3 by
cloning the 4.2 kb 3' ~I fragment into pBSl3+, digesting the resulting plasmid
with
~~HI and III, and circularizing by ligation the large fragment from the
digestion.
DG238 inserts EcoRV and sites immediately downstream of the TGA stop
colon. Mutant colony candidates were identified with [~2P]-labelled
oligonucleotide
DG239. Plasmid DNA isolated from positive colonies was screened for
appropriate




WO 92/03556 ~ ~ ~ ~ ~ ~ ~ PGT/US91/05753
,...
restriction digest patterns, and the DNA sequence was confirmed. Une correct
plasnud
obtained was designated as pTma3'mut#1 and later renamed pTma05.
B. Assembling the Full-Len~~h Gene in a lac Promoter Vector
For purposes of studying low level expression of ~ Pol I in ~. ~ and
5 possible complementation of ~. ~i polymerise mutants by r~r Pol I (where
high
level expression might kill the cell, but where low level expression might
rescue or
complement), the ~n Pol I gene was assembled in the pBS 13+ cloning vector. An
300 by ~I to EcoRV fragment from pTma3'mut#1 was isolated and purified,
following agarose gel electrophoresis and ethidium bromide staining, by
excising an
10 agarose gel slice containing the 300 by fragment and freezing in a Costar
spinex filter
unit. Upon thawing, the unit was spun in a microfuge, and the liquid
containing the
DNA fragment was collected. After ethanol precipitation, the fragment was
ligated with
each of the two 5'-mutated vectors, pTmaS'Nde#3 and pTmaS'Nco#9, which had
each
been digested with A~718, repaired with Klenow and all 4 dNTPs (the reaction
15 conditions are 56 mM Tris-Cl, pH 8.0, 56 mM NaCI, 6 mM MgCl2, 6 mM DTT, 5
~tM
dNTPs, and 11 units of Klenow at 3TC for 15 minutes; then inactivate at 75'C
for 10
minutes), and then further digested with Xm
The ligation was carried out in two steps. To ligate the sticky ends, the
conditions were 20 ~g/ml total DNA, 20 mM Tris-Cl, pH 7.4, 50 mM NaCI, 10 mM
20 MgCl2, 40 ~.M ATP, and 0.2 Weiss units T4 DNA ligase per 20 ~tl reaction at
0'C
overnight. To ligate X718-digested, Klenow repaired blunt ends with EcoRV-
digested blunt ends, the first ligations are diluted 4 to 5 fold and incubated
at 15'C in
the same ligation buffer, except 1 mM ATP and 10 Weiss units of T4 DNA ligase
are
used per 20 ~tl reaction. Ligations were transformed into DG101 host cells.
25 Candidates were screened for appropriate restriction sites, and the DNA
sequences
around the cloning sites was confirmed. The desired plasmids were designated
pTma08 (j~,~I site at ATG) and pTma09 ~I site at ATG).
C. Assembling the Full-Length Gene in P~l3xnression Vectors
The following table describes PL promoter expression vectors used for
30 assembling and expressing full-length ~ Pol I under the control of ~, PL
promoter.
Olieonucleotide
Duy~lexes


Vector Site RBS* AsuII+/-** ned into nDG160 61 Am
at ATG Clo or X rTn et***


pDG 174 ICI T7 - DG 106/DG 107 Amp


35 pDG 178 I~I N - DG 110/DG 111 Amp


pDG182 ~I T7 + FL42/FL,43 Amp


pDG184 ~I N + FL44/FL.45 Amp


pDG185 VII N + FL,44/F'L45 Tet






Wt~'t/03556 2 0 8 9 4 9 ~ p[T/L1S91/05753
46
"RBS - Phage T7 gene 10 or lambda gene N ribosome bind site.
** AsuII sites destroyed by digestion with ~.SI, repair with HIenow, and
ligation
of the repaired ends.
*** Antibiouc resistance determinant ampicillin or tetracycline.
The five vectors in the table are derivatives of plasmid pDG 160, if
ampicillin resistant,
or pDG161, if tetracycline resistant. Plasmids pDG160 and pDG161 and the
scheme
for constructing, vectors similar to the pDG vectors shown in the table are
described in
Patent Family A. The vectors confer ampicillin or tetracycline resistance and
all contain
the 8-toxin positive retroregulator from Bacillus thuringiensis and the same
point
mutations in the RNA II gene that render the plasmids temperature sensitive
for copy
number.
The probes and oligonucleotides described in the Table are shown below.
DG240 SEQ ID NO: 16
5'CCATCAAAAAGAAATAGTCTAGCCATATGTGTTTCCTGTGTGAAATTG
DG241 SEQ ID NO: 17 5'AAACACATATGGCTAGAC
DG244 SEQ ID NO: 18
5'CCATCAAAAAGAAATAGTCTAGCCATGGTTGTTTCCTGTGTGAAATTG
DG245 SEQ ID NO: 19 5'AAACAACCATGGCTAGAC
DG238 SEQ ID NO: 20
fGCAAAACATGGTCGTGATATCGGATCCGGAGGTGTTATCTGTGG
DG239 SEQ ID NO: 21 5'CCGATATCACGACCATG
DG106 SEQ ID NO: 22 5'CCGGAAGAAGGAGATATACATATGAGCT
DG107 SEQ ID NO: 23 5'CATATGTATATCTCCTTCTT '
DG110 SEQ ID NO: 24 5'CCGGAGGAGAAAACATATGAGCT
DGI11 SEQ ID NO: 25 5'CATATGTITTC'TCCT
FL42 SEQ ID NO: 26 5'CCGGAAGAAGGAGAAAATACCATGGGCCCGGTAC
FL43 SEQ ID NO: 27 5'CGGGCCCATGGTATTTTCTCCZTCT'T
FI,44 SEQ ID N0: 28 5'CCGGAGGAGAAAATCCATGGGCCCGGTAC
FL45 SEQ ID NO: 29 5'CGGGCCCATGGATTTTCTCCT
A three-fragment ligation was used to assemble the Tma Pol I gene in the
vectors. The vectors are digested with ~I and either NCI (pDG174, pDG178) or
NcoI (pDG 182, pDG 184, pDG 185). The 5' end of the r ~ Pol I gene is from
pTmaS'Nde#3 digested with N~igI and ~aI or pTmaS'Nco#9 digested with 1~c I and
Xmai. The 3' end of the gene is from pTma3'mut#1 digested with ~~I and EcoRV
and the -300 by fragment purified as described above.
The plasmid pDG 182 shown in the Table and the scheme above were used to
construct expression vector pTmal3. The plasmid pDG184 and the scheme above
were used to construct expression vectors pTmal2-1 and pTmal2-3. Plasmid
pTmal2-3 differs from pTmal2-1 in that pTmal2-3 is a dimer of.pTmal2-1
produced
B




WO 92/03556 ~ 0 $ 9 4 ~ ~ PCT/US91/05753
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47
during the same ligation/transformation protocol. The plasmid pDG185 and the
scheme
shown above were used to construct expression vector pTMal 1.
Even though a vector may contain the entire polymerise coding sequence, a
shortened form of the enzyme can be expressed either exclusively or in
combination
with a full length polymerise. These shortened forms of ~ DNA polymerise
result
from translation initiation occurring at one of the methionine (ATG) colons in
the
coding sequence other than the 5'-ATG. The monomeric pTmal2-1 plasmid
produces,
upon heat induction, predominantly a biologically active thermostable DNA
polymerise
lacking amino acids 1 through 139 of native ~ DNA polymerise. This
approximately 86 kDa protein is the result of translation initiation at the
r~thionine
colon at position 140 of the ~ coding sequence and is called MET140.
In shake flask studies under the appropriate conditions (heat induction at
34'C
or 36'C, but not 38'C), the multimeric pTmal2-3 expression vector yielded a
significant level of "full length" DNA polymerise (approximately 97 kDa by SDS-

PAGE) and a ::.naller amount of the shortened (approximately 86 kDa) form
resulting
from translation initiation at Met 140. Amino-acid sequencing of the full
length ~
DNA polymerise indicated that the amino-terniinal methionine was removed and
the
second-position alanine was present at the N-tcrminus.
Recombinant DNA Polymerise was purified from ~. ~ strain DG 116
containing plasmid pTmal2-3. The seed flask for a 10 L fermentation contained
tryntone (20 g/1), yeast extract (10 g/1), NaCI (10 g/1), ampicillin (100
mg/1), and
thiamine (10 mg/1). The seed flask was innoculated with a colony from an agar
plate (a
frozen glycerol culture can be used). The seed flask was grown at 30'C to
between 0.5
to 2.0 O.D. (A6~). The volume of seed culture inoculated into the ferznentor
is
calculated such that the bacterial concentration is 0.5 mg dry weight/liter.
The 12.5 liter
growth medium contained 60 mM K2HP04, 16 mM NaNla4HP04, 10 mM citric acid,
and 1 n.lM MgS04. The following sterile components were added: 2 g/1 glucose,
10
mg/1 thiamine, 2.5 g/1 casamino acids, 100 mg/1 ampicillin, and 100 mg/1
methicillin.
Foaming was controlled by the addition of propylene glycol as necessary, as an
antifoaming agent. Airflow was maintained at 21/min.
The fermentor was inoculated as described above, and the culture was grown at
30'C for 4.5 hours to a cell density (A68o) of 0.7. The growth temperature was
shifted
to 35'C to induce the synthesis orecombinant ~ DNA polymerise. The temperature
shift increases the copy number of the pTmal2-3 plasmid and simultaneously
derepresses the lambda PL promoter controlling transcription of the modified
Tma DNA
polymerise gene through inactivation of the temperature-sensitive cI repressor
encoded
by the defective pmphage lysogen in the host. The cells were grown for 21
hours to an
optical density of 4 (A68o) and harvested by centrifugation. The resulting
cell paste was
stored it -70' C.




WO 92/03556 PGT/US91/05753
48
Recombinant ~ DNA polymerise is purified as in Example 6, below.
Briefly, cells are thawed in 1 volume of TE buffer (50 mM Tris-Cl, pH 7.5, and
1.0
mM EDTA with 1mM DTT), and protease inhibitors are added (PMSF to 2.4 mM,
leupeptin to 1 ~tg/ml, and TLCK to 0.2 mM). The cells are lysed in an Aminco
french
pressure cell at 20,000 psi and sonicated to reduce viscosity. The sonicate is
diluted
with TE buffer and protease ,inhibitors to 5.5 X wet weight cell mass
(Fraction I),
adjusted to 0.3 M ammonium sulfate, and brought rapidly to 75'C and maintained
at
75'C for 15 min. The heat-treated supernatant is chilled rapidly to 0'C, and
the E_. ,~li'
cell membranes and dentaured proteins are removed following centrifugation at
20,000
X G for 30 min. The supernatant containing ~ DNA polymerise (Fraction II) is
saved. The level of Polymin P necessary to precipitate >95% of the nucleic
acids is
deternnined by trial precipitation (usually in the range of 0.6 to 1% w/v).
The desired
amount of Polymin P is added slowly with rapid stirring at 0'C for 30 min. and
the
suspension centrifuged at 20,000 X G for 30 min. to remove the precipitated
nucleic
acids. The supernatant (Fraction III) containing the DNA polymerise is saved.
Fraction III is applied to a phenyl separose column that has been equilibrated
in
50 mM Tris-Cl, pH 7.5, 0.3 M ammonium sulfate, 10 mM EDTA, and 1 mM DTT.
The column is washed with 2 to 4 column volumes of the same buffer (A~o to
baseline), and then 1 to 2 column volumes of TE buffer containing 100 mM KCI
to
remove most contaminating ~. ~1' proteins. Tma DNA polymerise is then eluted
from
the column with buffer containing 50 mM Tris-Cl, pH 7.5, 2 M urea, 20% (w/v)
ethylene glycol, 10 mM EDTA, and 1 mM DTT, and fractions containing DNA
polymerise activity are pooled (Fraction IV).
Final purification of recombinant Tma DNA polymerise is achieved using
heparin sepharose chromatography (as for native or MET284 recombinant DNA
polymerise), anion exchange chromatography, or affigel blue chromatography.
Recombinant Tma DNA polymerise may be diafiltered into 2.SX storage buffer,
combined with 1.5 volumes of sterile 80% (w/v) glycerol, and stored at -
20°C.
Expression of a Truncated Tma Polymerise MET284
As noted above, expression plasmids containing the complete ~n gene coding
sequence expressed either a full length polymerise resulting from translation
initiation
at the start codon or a shortened polymerise resulting from translation
initiation
occurring at the methionine codon at position 140. A third methionine codon
that can
act as a translation initiation site occurs at position 284 of the Tma gene
coding
sequence. Plasmids that express a DNA polymerise lacking amino acids 1 through
283
of native ~ DNA polymerise were constructed by introducing deleting
corresponding regions of the Tma coding sequence.




WO 92/03556 PGT/US91/05753
~' 2089495
49
Plasmid pTmal2-1 was digested with BsuHl (nucleotide position 848) and
indIl? (nucleotide position 2629). A 1781 base pair fragment was isolated by
agarose
gel purification. To separate the agarose from the DNA, a gel slice containing
the
desired fragment was frozen at -20'C in a Costar spinex filter unit. After
thawing at
room temperature, the unit was spun in a microfuge. The filtrate containing
the DNA
was concentrated in a Speed Vac concentrator, and the DNA was precipitated
with
ethanol.
The isolated fragment was cloned into plasmid pTmal2-1 digested with T~I
and ~;dIII. Because ~I digestion leaves the same cohesive end sequence as
digestion with 1, the 1781 base pair fragment has the same cohesive ends as
the
full length fragment excised from plasmid pTmal2-1 by digestion with ~I and
it dIII. The ligation of the isolated fragment with the digested plasmid
results in a
fragment switch and was used to create a plasmid designated pTmal4.
Plasmid pTmalS was similarly constructed by cloning the same isolated
fragment into pTmal3. As with pTmal4, pTmalS drives expression of a polymerise
that lacks amino acids 1 through 283 of native ~ DNA polymerise; translation
initiates at the methionine colon at position 284 of the native coding
sequence.
Both the pTmal4 and pTmalS expression plasmids expressed at a high level a
biologically active thermostable DNA polymerise of molecular weight of about
70 kDa;
plasmid pTmalS expressed polymerise at a higher level than did pTmal4. Based
on
similarities with ~. ~(i Pol I Klenow fragment, such as conservation of amino
acid
sequence motifs in all three domains that are critical for 3'-5' exonuclease
activity,
distance from the amino terminus to the first domain critical for exonuclease
activity,
and length of the expressed protein, the shortened form (ME'T284) of Tma
polymeras°
should possess 3'-5' exonuclease and proof reading activity but lack 5'-3'
exonuclease
activity. However, initial SDS activity gcl assays and solution assays for 3'-
5'
exonuclease activity suggested significant attenuation in the proof reading
activity of the
polymerise expressed by ~. ~ host cells harboring plasmid pTmalS.
MET284 ~ DNA Polymerise was purified from ~. ~ strain DG 116
containing plasmid pTmalS. The seed flask for a 10 L fermentation contained
tryptone
(20 g/1), yeast extract ( 10 g/1), NaCI ( 10 g/1), glucose ( 10 g/1),
ampicillin (50 mg/1), and
thiamine ( 10 mg/1). The seed flask was innoculated with a colony from an agar
plate (a
frozen glycerol culture can be used). The seed flask was grown at 30'C to
between 0.5
to 2.0 O.D. (A6go). The volume of seed culture inoculated into the fermentor
is
calculated such that the bacterial concentration is 0.5 mg dry weight/liter.
The 10 liter
growth medium contained 25 mM KH2P04, 10 mM (NH4)2S04, 4 mM sodium citrate,
0.4 mM FeCl3, 0.04 mM ZnCl2, 0.03 mM CoCl2, 0.03 mM CuCl2, and 0.03 mM
H3B03. The following sterile components were added: 4 mM MgS04, 20 g/1
glucose,
20 mg/1 thiamine, and 50 mg/1 ampicillin. The pH was adjusted to 6.8 with NaOH
and




WO 92/03556 PCT/US91/OS753
2Q8~4~5 50
controlled during the fermentation by added NH40H. Glucose was continually
added
by coupling to NH40H addition. Foaming was controlled by the addition of
propylene
glycol as necessary, as an antifoaming agent. Dissolved oxygen concentration
was
maintained at 40%.
The fermentor was inoculated as described above, and the culture was grown at
30°C to a cell density of 0.5 to 1.0 X lOlo cells/ml (optical density
[A~] of 15). The
growth temperature was shifted to 38°C to induce the synthesis of MH
TT284 ~ DNA
polymerise. The temperature shift increases the copy number of the pTmalS
plasmid
and simultaneously derepresses the lambda PL promoter controlling
transcription of the
modified ~ DNA polymerise gene through inactivation of the temperature-
sensitive
cI repressor encoded by the defective prophage lysogen in the host.
The cells were grown for 6 hours to an optical density of 37 (A~o) and
harvested by centrifugation. The cell mass (ca. 95 g/1) was resuspended in an
equivalent volume of buffer containing 50 mM Tris-Cl, pH 7.6, 20 mM EDTA and
20% (w/v) glycerol. The suspension was slowly dripped into liquid nitrogen to
freeze
the suspension as "beads" or small pellets. The frozen cells were stored at -
70°C.
To 200 g of frozen beads (containing 100 g wet weight cell) were added 100 ml
of 1X TE (50 mM Tris-Cl, pH 7.5, 10 mM EDTA) and DTT to 0.3 mM, PMSF to 2.4
mM, leupeptin to 1 ~.g/ml and TLCK (a protease inhibitor) to 0.2 mM. The
sample
was thawed on ice and uniformly resuspended in a blender at low speed. The
cell
suspension was lysed in an Aminco french pressure cell at 20,000 psi. To
reduce
viscosity, the lysed cell sample was sonicated 4 times for 3 min. each at
50°!o duty cycle
and 70% output. The sonicate was adjusted to 550 ml with 1X TE containing 1 mM
DTT, 2.4 mM PMSF, 1 ~tg/ml leupeptin and 0.2 mM TLCK (Fraction I). After
addition of ammonium sulfate to 0.3 M, the crude lysate was rapidly brought to
75°C in
a boiling water bath and transferred to a 75°C water bath for 15 min.
to denature and
inactivate ~. ~ host proteins. The heat-treated sample was chilled rapidly to
0°C and
incubated on ice for 20 min. Precipitated proteins and cell membranes were
removed
by centrifugation at 20,000 X G for 30 min. at 5°C and the supernatant
(Fraction II)
saved.
The heat-treated supernatant (Fraction II) was treated with polyethyleneimine
(PEI) to remove most of the DNA and RNA. Polymin P (34.96 ml of 10% [w/v], pH
7.5) was slowly added to 437 ml of Fraction II at 0°C while stirring
rapidly. After 30
min. at 0°C, the sample was centrifuged at 20,000 X G for 30 min. The
supernatant
(Fraction III) was applied at 80 ml/hr to a 100 ml phenylseparose column
(3.2x12.5
cm) that had been equilibrated in 50 mM Tris-Cl, pH 7.5, 0.3 M ammonium
sulfate, 10
mM EDTA, and 1 mM DTT. The column was washed with about 200 ml of the same
buffer (A~o to baseline) and then with 150 ml of 50 mM Tris-Cl, pH 7.5, 100 mM
KCI, 10 mM EDTA and 1 mM DTT. The MET284 Tma DNA polymerise was then




WO 92/03556 PGT/US91105753
20~940~
51
eluted from the column with buffer containing 50 mM Tris-C1, pH 7.5, 2 M urea,
20%
(w/v) ethylene glycol, 10 mM EDTA, and 1 mM DTT, and fractions containing DNA
polymerise activity were pooled (Fraction IV).
Fraction IV is adjusted to a conductivity equivalent to 50 mM KCl in 50 mM
Tris-Cl, pH 7.5, 1 mM EDTA, and 1 mM DTT. The sample was applied (at 9 ml/hr)
to
a 15 ml heparin-sephamse column that had been equilibrated in the same buffer.
The
column was washed with the same buffer at ca. 14 ml/hr (3.5 column volumes)
and
eluted with a 150 ml 0.05 to 0.5 M KCl gradient in the same buffer. The DNA
polymerise activity eluted between 0.11-0.22 M KCI. Fractions containing the
pTmalS encoded modifed ~ DNA polymerise are pooled, concentrated, and
diafiltered against 2.5X storage buffer (50 mM Tris-Cl, pH 8.0, 250 mM KCI,
0.25
mM EDTA, 2.5 mM DTT, and 0.5% Tween 20), subsequently mixed with 1.5
volumes of sterile 80% (w/v) glycerol, and stored at -20'C. Optionally, the
heparin
sepharose-eluted DNA polymerise or the phenyl sepharose-eluted DNA polymerise
can be dialyzed or adjusted to a conductivity equivalent to 50 mM KCl in 50 mM
Tris-Cl, pH 7.5, 1 mM DTT, 1 mM EDTA, and 0.2% Tween 20 and applied ( 1 mg
protein/ml resin) to an affigel blue column that has been equilibrated in the
same buffer.
The column is washed with three to five column volumes of the same buffer and
eluted
with a 10 column volume KCl gradient (0.05 to 0.8 M) in the same buffer.
Fractions
containing DNA polymerise activity (eluting between 0.25 and 0.4 M KCl) are
pooled,
concentrated, diafiltercd, and stored as above.
The relative thermoresistance of various DNA polymerises has been compared.
At 97.5'C the half life of native ~ DNA polymerise is more than twice the half
life
of either native or recombinant T~,a DNA (i.e., AmpliTaq~) DNA polymerise.
Surprisingly, the half life at 97.5'C of MET284 DNA polymerise is 2.5 to 3
times
longer than the half life of native ~ DNA polymerise.
PCR tubes containing 10 mM Tris-Cl, pH 8.3, and 1.5 mM MgCl2 (for T~ or
native ~ DNA polymerise) or 3 mM MgCl2 (for MET284 ~ DNA polymerise),
50 mM KCl (for ~, native T~ and MET284 ~ DNA polymerises) or no KCl (for
MET284 ~ DNA polymerise), 0.5 ~t.M each of primers PCRO1 and PCR02, 1 ng of
lambda template DNA, 200 ~,M of each dNTP except dCTP, and 4 units of each
enzyme were incubated at 97.5°C in a large water bath for times ranging
from 0 to 60
min. Samples were withdrawn with time, stored at 0°C, and 5 ~1 assayed
at 75°C for
10 min. in i stindard activity assay for residual activity.
~ DNA polymerise had a half life of about 10 min. at 97.5'C, while native
DNA polymerise had a half life of about 21 to 22 min. at 97.5'C. Surprisingly,
the MET284 form of DNA polymerise had a significanlty longer half life (50 to
55 min.) than either ~ or native Tma DNA polymerise. The improved
therrnoresistance of MET284 Tma DNA polymerise will find applications in PCR,




WO 92/03556 PGT/US91/05753
2(1~~~~~~ s2
particularly where G+C-rich targets are difficult to amplify because the
strand-separation temperature required for complete denaturation of target and
PCR
product sequences leads to enzyme inactivation.
PCR tubes containing 50 ~tl of 10 mM Tris-Cl, pH 8.3, 3 mM MgCl2, 200 ~M
of each dNTP, 0.5 ng bacteriophage lambda DNA, 0.5 ~M of primer PCRO1, 4 units
of MET284 Tma DNA polymerase, and 0.5 ~tM of primer PCR02 or PL10 were cycled
for 25 cycles using T~" of 96°C for 1 min. and T~,~~_~~,d of
60°C for 2 min.
Lambda DNA template, deoxynucleotide stock solutions, and primers PCRO1 and
PCR02 were part of the PECI GeneAmp~ kit. Primer PL10 has the sequence: (SEQ
ID NO. 45) 5'-GGCGTACCTTTGTCTCACGGGCAAC-3' and is complementary to
bacteriophage lambda nucleotides 8106-8130.
The primers PCRO1 and PCR02 amplify a 500 by product from lambda. The
primer pair PCRO1 and PL10 amplify a 1 kb product from lambda. After
amplification
with the respective primer sets, 5 ~t.l aliquots were subjected to agarose gel
is elecaophoresis and the specific intended product bands visualized with
ethidium
bromide staining. Abundant levels of product were generated with both primer
sets,
showing that MET284 ~ DNA polymerase successfully amplified the intended
target
sequence.
Exml
Expression of Truncated Tma Polymerase
As noted above, host ells transformed with plasmids that contain the complete
~ DNA polymerase gene coding sequence express a shortened form (MET140) of
Tma polymerise either exclusively or along with the full length polymerise.
Mutations
can be made to control which form of the polymerise is expressed. To enhance
the
exclusive expression of the MET140 form of the polymerise, the coding region
corresponding to amino acids through 139 were deleted from the expression
vector.
The protocol for constructing such a deletion is similar to the construction
described in
Example 6: a shortened gene fragment is excised and then reinserted into a
vector from
which a full length fragment has been excised. However, the shortened fragment
can
be obtained as a PCR amplification product rather than purified from a
restriction
digest. This methodology allows a new upstream restriction site (or other
sequences)
to be incorporated where useful.
To delete the region up to the methionine codon at position 140, an ,~hI site
was introduced into pTmal2-1 and pTmal3 using PCR. A forward primer (FL63) was
designed to introduce the S~hI site just upstream of the methionine codon at
position
140. The reverse primer (FL69) was chosen to include an X~I at position 624.
Plasmid pTmal2-1 linearized with,~maI was used as the PCR template, yielding a
22s
by PCR product.




WO 92/03556 PCT/US91/05753
2089495
53
Before digestion, the PCR product was treated with 50 ~g/ml of Proteinase K
in PCR reaction mix plus 0.5% SDS and 5 mM EDTA. After incubating for 30
minutes at 37'C, the Proteinase K was heat inactivated at 68'C for 10 minutes.
This
procedure eliminated any T~ polymerase bound to the product that could inhibit
subsequent restriction digests. The buffer was changed to a TE buffer, and the
excess
PCR primers were removed with a Ccntricon 100 microconcentrator.
The amplified fragment was digested wah ~I, then treated with Klenow to
create a blunt end at the ~1 I-cleaved end, and finally digested with ~I. The
resulting fragment was ligated with plasmid pTtnal3 (pTmal2-1 would have been
s 10 suitable) that had been digested with'j~I, repaired with Klenow, and then
digested
with ~I. The ligation yielded an in-frame coding.sequence with the region
between
the initial NcoI site (upstream of the first methionine codon of the coding
sequence) and
the introduced S~h_I site (upstream of the methionine codon at position 140)
deleted.
The resulting expression vector was designated pTmal6.
The primers used in this example are given below.
Primer ~,QID NO: Seauence
FL63 SEQ ID NO: 30 5'GATAAAGGCATGCTTCAGCTTGTGAACG
FL69 SEQ ID NO: 31 5'TGTACTTCTCTAGAAGCTGAACAGCAG
Ex m 1
Fiiminatio_n_ of lnr~eti ri RRS in MET140 Exnrescion Vectors
Reduced expression of the MET140 form of ~ DNA polymerise can be
achieved by eliminating the ribosome binding site (RBS) upsatam of the
methioninc
codon at position 140. The RBS was be eliminated via oligonucleotide site-
directed
mutagenesis without changing the amino acid sequence. Taking advantage of the
redundancy of the genetic code, one can make changes in the third position of
codons
to alter the nucleic acid sequence, thereby eliminating the RBS, without
changing the
amino acid sequence of the encoded protein.
A mutagenic primer (FT.64) containing the modified sequence was synthesized
and phosphor;dated. Single-stranded pTma09 (a full length clone having an 1~I
site)
was prepared by coinfecting with the helper phage 8408, commercially available
from
Stra:agene. A "gapped duplex" of single stranded pTma09 and the large fragment
from
the '~v II digestion of pBS 13+ was created by mixing the two plasmids,
heating to
boiling for 2 minutes, and cooling to 65'C for 5 minutes. The phosphorylated
primer
was then annealed with the "gapped duplex" by mixing, heating to 80'C for 2
minutes,
and then cooling slowly to room temperature. The remaining gaps were filled by
extension with Klcnow and the fragments ligatcd with T4 DNA ligase, both
reactions
B




W,,A92/03556 2 p 8 9 ~ 9 5 PCT/US91/05753
54
taking place in 200 ~tM of each dNTP and 40 ~tM ATP in standard salts at 37'C
for 30
minutes.
The resulting circular fragment was transformed, into DG 101 host cells by
plate
transformations on nitrocellulose filters. Duplicate filters were made and the
presence
of the correct plasmid was detected by probing with a y32P-phosphorylated
probe
(FL6$). The vector chat resulted was designated pTmal9.
The RBS minus portion from pTmal9 was cloned into pTmal2-1 via an
NcoI/~t I fragment switch. Plasmid pTmal9 was digested with , 1~I and ~t I,
and
the 620 by fragment was purified by gel electrophoresis, as in Example 7,
above.
Plasmid pTmal2-1 was digested with VII, ~I, and ~I. The ~I cleavage
inactivates the RBS+ fragment for the subsequent ligation step, which is done
under
conditions suitable for ligating "sticky" ends (dilute ligase and 40 EtM ATP).
Finally,
the ligation product is transformed into DG 116 host cells for expression and
designated
pTmal9-RBS.
1$ The oligonucleoade sequences used in this example are listed below-
Q~gQ SEQ ID NO: ,,tee uen,~
FL64 SEQ ID NO: 32 $'CTGAAGCATGTCTITGTCACCGGTTACTATGAATAT
FL6$ SEQ ID NO: 33 $'TAGTAACCGGTGACAAAG
xam 1
To effect translation initiation at about the aspartic acid colon at position
21 of
the Tma DNA polymerise gene coding sequence, a methionine colon is introduced
before the colon, and the region from the initial ~I site to this introduced
methionine
colon is deleted. The deletion process involves FCR with the same downstream
primer described about (FL69) and with an upstream primer (FL66) designed to
incorporate an 1~I site and a methionine colon to yield a $70 base pair
product.
The amplified product is concentrated with a Centricon-100 microconcentrator
to eliminate excess primers and bu:fer. The product is concentrated in a Speed
Vac ""'
concentrator and then resuspended in the digestion mix. The amplified product
is
digested with coI and Xt~I. Likewise, pTmal2-1, pTmal3, or pTmal9-RBS is
digested with the same two restriction enzymes, and the digested, amplified
fragment is
ligated with the digested expression vector. The resulting construct his a
deletion from
the NCI site upstream of the start colon of the native ~ coding sequence to
the new
methionine colon introduced upstream of the aspardc acid colon at position 21
of the
native Tma coding sequence.
Similarly, a deletion mutant can be created such that translation initiation
begins
at G1u74, the ~lutamic acid colon at position 74 of the native Tma coding
seouence.
B




W(~/03556 2 p g 9 4 9 5 PCT/US91/05753
An upstream primer (FL67) is designed to introduce a methionine colon and an
NcoI
site before G1u74. The downstream primer and cloning protocol used are as
described
above for the MET-ASP21 construct.
The upstream primer sequences used in this example are listed below.
S
0~~.~o SEO ID NO: a uence
FL66 SEQ ID NO: 34 5'CTATGCCATGGATAGATCGCTTTCTACTTCC
FL67 SEQ ID NO: 35 5'CAAGCCCATGGAAACTTACAAGGCTCAAAGA
xam 1
10 ~xnression Vectors With T7 Promoters
Expression efficiency can be altered by changing the promoter and/or ribosomal
binding site (RBS) in an expression vector. The T7 GenelO promoter and RBS
have
been used to drive expression of DNA polymerise from expression vector
pTmal7, and the T7 GenelO promoter and the Gene N RBS have been used tn drive
15 expression of ~ DNA polymerise from expression vector pTmal8. The
construction of these vectors took advantage of unique restriction sites
present in
pTmal2-1: an I~flII site upstream of the promoter, an ICI site downstream of
the
RBS, and a B~,~I site between the promoter and the RBS. The existing promoter
was _
excised from pTmal2-1 and replaced with a synthetic T7 Gene 10 promoter using -

20 techniques similar to those described in the p: cvious examples.
The synthetic insert was created from two overlapping synthetic
oligonucleotides. To create pTmal7 (with T7 Gene 10 RBS), equal portions of
FR414
and FR416 were mixed, heated to boiling, and cooled slowly to room
temperature.
The hybridized oligonucleotides were extended with Klenow to create a full
length
25 double stranded insert. The extended fragment was then digested with ~fIII
and I~I,
leaving the appropriate "sticky" ends. The insert was cloned into plasmid
pTmal2-1
digested with ~(II and 1~I. DG 116 host cells were transformed v; ith the
resulting
plasmid and transformanu screened for the desired plasm:~~ .
The same procedure was used in the creation of p i~mal8 (with Gene N RBS),
30 except that FR414 and FR418 were used, and the extended fragment was
digested with
AflII and B,~EI. This DNA fragment was substituted for the P~ promoter in
plasmid
pTmal2-1 that had been digested with A~III and _B~EI.
Plasmids pTmal7 and pTmal8 are used to transform ~. ~ host cells that have
been modified to contain an inducible T7 DNA polymerise gene.
35 The oligonucleorides used in the construction of these vectors are listed
below.
B




W~''2/03556 ~ ~ 8 9 ~ 9 5 PGT/US91/05753
56
FR414 SEQ 117 NO: 36
S'TCAGCTTAAGACTTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTT-
CCCTC
FR416 SEQ )D NO: 37
S 5'TCGACCATGGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGAAACC-
GTTG
FR418 SEQ ID NO: 38
S'TCAGTCCGGATAAACAAAATTATTTCTAGAGGGAAACCGTTG
Example 11
Translational Cou In inQ
As described about, translational coupling can increase the efficiency of
expression of a protein by coupling a short coding sequence just upstream of
the
initiation site of the coding sequence for the pmtein. Termination of
translation of the
upstream coding sequence leaves the ribosome in close proximity to the
initiation site
for the downstream coding sequence. The upstream coding sequence functions
only to
move the ribosome downstream to the start of the coding sequence for the
desired
protein.
Translationally coupled ~ expression vectors were constructed with the
translation initiation signal and first ten colons of the T7 bactcriophage
major capsid
protein (gene 10) fused in-frame to the last six colons of ~. ~1'~ TrpE placed
upstream
of the ~ coding region. The TGA (stop) colon for TrpE is "coupled" with the
ATG
(start) colon for the ~ gene, forming the sequence TGATG. A one base flame-
shift
is required between translation of the short coding sequence and translation
of the Tma
coding sequence.
In the example below, a fragment containing the T7 Gene 10-~. ~ TrpEITrpD
fusion product (the last 6 colons and TGA stop colon from TrpE along with the
overlapping ATG start colon from TrpD) was transferred from a pre-existing
plasmid.
One of ordinary skill will recognize that the T7 Gene 10-~. ~ TrpE~I'rpD
fusion
product used in the construction of the transladonally coupled expression
vectors can be
constructed as a synthetic oligonucleotide. The sequence for the inserted
fragment is
listed below.
The T7 Gene 10-E. c2li TrpE~I"rpD fusion product was amplified from plasmid
pSYC1868 with primers FL.48 and FL49. With primers FL51 and FL53, the 5' end
of
the ~ Pol I gene in pTma08 (a full length clone containing an 1~I site) was
amplified from the ATG start colon to the MroI site downstream of the ATG
start
colon. The primers FL51 and FL49 were designed to leave overlapping regions
such
that the two amplified products could be annealed and extended, essentially as
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2089495
s7
described in Example 10. The two amplification products were mixed, heaed to
95'C,
slowly cooled to room temperature to anneal, and extended w ith ~aq
polymerase.
the extended insert v: as amplified with primers FL48 and FL53 and then
digested with ~I and ~I. Plasmid pTmal2-1 was digested with ~I and treated
with calf intestine alkaline phosphatase to prevent re-ligation. The digested
pTmal2-1
was ligated with the insert. DG 116 host cells were transformed with the
resulting
construct and transformants screened for the desired plasmid DNA. The
resulting
vector was designated pTma20.
The soqucnces of the oligonucleotide primers and the T7 Gene 10-~. ~f
TrpEn'rpD fusion product (Gene 10 insert) arc listed below.
'm r SEO m NO: Seaue~c


FL48 SEQ ID NO: 5'TCCGGACTITAAGAAGGAGATATAC
39


FL49 SEQ ID NO: S'AATAGTCTAGCCATCAGAAAGTCTCCTGTGC
40


IS FL51 SEQ )D NO: 5'AGACTZTCTGATGGCTAGACTATTTCIT _
41


FL53 SEQ ID NO: 5'CTGAATCAGGAGACCCGGGGTCTTTGGTC -
42


GenelO insert SEQ B7 NO:
s'CTTTAAGAAGGAGATATACATATGGCTAGCATGACTGGTGGACAGCAAATG
CATGCACAGGA~ACTTTCT~ATG
Exam I
Arg U tRNA Ex rcssion
The pattern of codon usage differs between Thermotoga g~jg~~ and ~. ~.
In the Tma coding sequence, arginine is most frequently coded for by the "AGA"
codon, whereas this codon is used in low_ frequency in ~. ~ host cells. The
corresponding "Arg U" tRNA appears in low concentrations in ~. ~I't. The low
concentration in the host cell of Arg tRNA using the "AGA" codon may limit the
translation efficiency of the ~ polymerase gene. The eff ciency of translation
of the
~ coding sequence within an ~. ~ host may be improved by increasing the
concenaation of this tRNA species by cloning multiple copies of the tRNA gene
into
the host cell using a second expression vector that contains the gene for the
"Arg U"
tRNA.
The Arg U tRNA gene was PCR amplified from _E. Eli genomic DNA using
the primers DG284 and DG286. The amplification product was digested with ~I
and
~~HI. The ~,IEI compatible vector pACYC184 was digested with ~I and ~FiI,
and the Arg U gene fragment was subsequently ligated with the digested vector.
DG 101 cells were transformed, and the ligated vector was designated pARG0l.
Finally, DG116 host cells were co-transformed with pARG01 and pTmal2-1.
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The oligonucleotide primers used in this Example are listed below
Prim r SEO )17 NO: ~gg~e_nce
DG284 SEQ >D NO: 43 5'CGGGGATCCAAAAGCCATTGACTCAGCAAGG
DG285 SEQ 1D NO: 44 5'GGGGGTCGACGCATGCGAGGAAAATAGACG
B