Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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X-6125C-Canada -l-
VECTORS FOR EXPRESS ING
BOVINE GROWTH HORMONE DERIVATIVES
The present invention relates to a novel
selectable and autonomously replicating recombinant DNA
expression vector which comprises 1) a transcriptional
and translational activating nucleotide sequence which
is in the reading frame of a gene that codes or a
bioactive derivative of bovine growth hormone (bGH) and
2) a replicon which, under induced conditions, loses
copy number control. The invention further relates to
novel transformants of the aforementioned vectors, bovine
growth hormone derivatives and methods of use.
The development and exploitation of recom-
binant DNA technoloqy for the production of bGH has
been severely handicapped by problems of gene expression.
Some of these problems, detailed in European Patent
Application Publication No. 0075444, involve the tran-
scription of functionally suboptimal mRNA. Such mRNA
has stem and loop secondary configurations that
~ap
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X-6125C-Canada -2-
interfere ~ith normal ribosome binding and thus drasti-
cally reduce or even prevent bGH expression. The
present invention circumvents this problem by providing
vectors for the high level expression of bGH
derivatives.
For purposes of the present invention as
disclosed and claimed herein, the following terms are as
defined below.
Recombinant DNA Cloning Vector - any autono-
mously replicating agent, including but not limited toplasmids, comprising a DNA molecule to which one or more
additional DNA segments can be or have been added.
Recombinant DNA Expression Vector - any
recombinant DNA cloning vector into which one or more
transcriptional and translational activator sequence(s)
have been incorporated.
Transcriptional Activating Sequence - any DNA
sequence that directs or provides for the transcription
of DNA into a mRNA transcript,
Translational Activating Sequence - any DNA
sequence that directs or provides for the translation of
a m~NA transcript into a peptide or polypeptide.
Translational Start Signal - any DNA triplet
that codes for a translational start codon.
Translational Stop Signal - any DNA triplet
that codes for a translational stop codon.
Transformation - the introduction of DNA into
a recipient host cell that changes the genotype.
Transformant - a recipient host cell that has
undergone transformation.
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X-6125C-Canada 3
~ estriction Fragment - any linear DNA sequence
generated by the action of one or more restriction
enzymes.
Replicon - any DNA sequence that controls the
replication of recombinant DNA cloning and expression
vectors.
Runaway Replicon - a replicon which lacks or
can be induced to lose copy number control, such loss
resulting in the uncontrolled replication and an extreme
increase in the copy number of DNA into which such
replicon has been incorporated.
Functional Polypeptide - a recoverable
bioactive heterologous polypeptide or precursor, a
recoverable bioactive polypeptide comprising a
heterologous polypeptide and a portion or whole of a
homologous polypeptide, or a recoverable bioinactive
fusion polypeptide comprising a heterologous polypeptide
and a bioinactivating polypeptide which can be specifi-
cally cleaved.
Fused Gene Product - a recoverable
heterologous polypeptide which comprises a portion or
whole of a homologous polypeptide.
Descri~tion Of The Fiqures
Figures 1-4 - Schematic illustration of the
construction protocol for plasmid pNM575.
F:~gures 5-8 - Schematic illustration of the
construction protocol for plasmid pNM789B.
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X-6125C-Canada
Figure 9 - Restriction site map of plasmids
pCZl920 and pJR1.
Figure lO - Restriction site map of plasmid
pCZ112.
Figure 11 - Thymosin alpha l synthetic gene.
Figure 12 - Synthesis protocol for nucleotide
fragment Tl5.
Figure 13 - Construction protocol for
plasmid pTh~l.
Figure 14 - ~roinsulin syntheti,c gene.
Figure 15 - Construction protocol for
plasmid pHI7~4~1.
Figure 16 - Construction protocol for
plasmid pHI104.
Detailed Description Of The Invention
The present invention provides, in one aspect, a
selectable and autonomously replicating recombinant DNA
expression vector which comprises
a) a runaway replicon and
b~ a tra~scriptional and translational activating
sequence which is in the reading frame of a
gene that codes for a bioactive bovine growth
hormone derivative, said gene being
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X-6125C-Canada _5_
(1) 5' ATG AAA GGG AAT TCT ATG GCC TTC
,- ... ... ... ... .., ... ,..
3' TAC TTT CCC TTA AGA TAC CGG AAG
CCA GCC ATG TCC TTG TCC GGC 3'
rI I I, ~ R-Rl
GGT CGG TAC AGG AAC AGG CCG 5',
(2) S' ATG GAT GAT AAG TTT CCG GCT ATG
1 r, . t I I I I I I . I . I I I -
3' TAC CTA CTA TTC AAA GGC CGA TAC
TCT CTG TCC GGC 3'
III III III III_O_Dl
AGA GAC AGG CCG A A 5~,
(3) 5' ATG TTT CCA GCC ATG GCT CTA
-- -- -- -- I- ~, ..
3' TAC AAA GGT CGG TAC CGA GAT
TCT GGT _ 1 3'
AGA CCA R R 5~,
(4) 5' ATG TTC CCA GCT ATG TCT CTA
~ ,., ,,, ,,, ,,, , " ".
3' TAC AAG GGT CGA TAC AGA GAT
TCT GGT_ 1 3'
AGA CCA R R 5"
(5) 5' ATG GAT TTT CCG GCT ATG TCT
... ,.. ... ... ... .,. ...
3' TAC CTA AAA GGC CGA TAC AGA
CTG TCC GGC 1 3'
I I I I I I I I I _D_D
GAC AGG CCG ~ ~ 5',
(6) 5' ATG TTT CCA GCT ATG TCT CTA
... ... ... ... ... -. .-I
3' TAC AAA GGT CGA TAC AGA GAT
TCT GGT_ _ 1 3'
AGA CCA R R 5,
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X-6125C-Canada -6-
(7) 5' ATG GTT TTT CCG GCT ATG TCT
... ... ... ... ... ... ...
3' TAC CAA AAA GGC CGA TAC AGA
CTG TCC GGC 1 3'
GAC AGG CcG~R~R 5,
or
(8) 5' ATG GCT TTT CCG GCT ATG TCT
... ... ... ... ... ... ...
3' TAC CGA AAA GGC CGA TAC AGA
CTG TCC GTC 1 3'
GAC AGG CAG R-R 5,
wherein
A is deoxyadenyl,
G is deoxyguanyl,
C is deoxycytidyl,
T is thymidyl,
R is a DNA sequence that codes for amino acids 9
(leucine) through 191 (phenylalanine) of bovine growth
hormone, and
Rl is a DNA seguence that codes for a translational
stop signal which is
5' TAA 3' 5' TAG 3'
... ...
3' ATT 5', 3' ATC 5', or
5' TGA 3'
3l ACT 5'
The invention further pr~vides novel transformants of
the aforementioned vectors, compounds, granules and
methods of use.
The above-recited aspect of the present invention
is also disclosed, and is claimed, in Canadian Patent Application
No. 475,731, filed March 5, 1985, of which this application is a
divisional.
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The present invention can be constructed by
independently ligating the following XbaI-HqiAI DNA
linker sequences
a) 5' CTAGAGGGTATTAATA ATG AAA GGG AAT TCT ATG
TCCCATAATTAT TAC TTT CCC TTA AGA TAC
GCC TTC CCA GCC ATG TCC TTG TCC G&C CTG
... ... ... ... ... ... ... ... ... ...
CGG AAG GGT CGG TAC AGG AAC AGG CCG GAC
TTT GCC AAC GCT GTGCT 3'
... ... ... ... .
AAA CGG TTG CGA C 5',
b) S' CTAGAGGGTATTAATA ATG TTT CCA GCC ATG GCT
........ -... ... ... ... ... -- --
3' TCCCATAATTAT TAC AAA GGT CGG TAC CGA
CTA TCT GGT CTG TTT GCC AAC GCT GTGCT 3'
... ... ... ... ... ... ... .-- -
GAT AGA CCA GAC AAA CGG TTG CGA C 5'
c) S' CTAGAGGGTATTAATA ATG TTT CCA GCT ATG TCT
............ ... ... ... ... ... ...
3' TCCCATAATTAT TAC AAA GGT CGA TAC AGA
CTA TCT GGT CTG TTT GCC AAC GCT GTGCT 3'
... ... ... ... ... ... ... ... .
GAT AGA CCA GAC AAA CGG TTG CGA C 5',
into the ~10.2 kb BamHI-XbaI and ~0.6 ~b BamHI-HqiAI
fragments of plasmid pCZ101. The resultant plasmids,
respectively designated as plasmids pCZ1920, pJRl and
pAT2, contain 1) the E. coli lipoprotein gene tran-
scriptional and translational activating se~uence
(Nakamura and Inouye, 1979, Cell 18:1109) in the reading
frame of a coding sequence for a bioactive bovine growth
hormone derivative; 2) an appropriately placed trans-
lational stop signal; and 3) a runaway replicon. Cells
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X-6125C-Canada -8-
transformed by plasmids pCZ1920, pJRl and pAT2 respec-
tively express MET LYS-GLY-ASN-SER-MET-ALA-bG~,
MET-PHE-PRO-ALA-MET-ALA-R2 and MET-bG~ wherein
MET is methionine,
LYS is lysine,
GLY is glycine,
ASN is asparagine,
SER is serine,
ALA is alanine,
PEE is phenylalanine,
PRO is proline,
bGH is the natural amino acid sequence of bovine
growth hormone beginning with the N-terminal (first)
phenyl al anine
R is the natural amino acid sequence of bovine
growth hormone beginning with the sixth amino acid
(leucine) from the N-terminus.
A restriction site map of each of plasmids pCZ1920 and
pJR1 i& presented in Figure 9 of the accompanying
drawings.
Additional plasmids that are also within the
scope of the present invention can be constructed by
independently ligating the following _~gI-HqiAI DNA
linker sequences
a) 5' CGACC ATG GAT GAT AAG TTT CCG GCT ATG TCT
,...... ... ... ... ... ... ... ... ... ... ...
TGG TAC CTA CTA TTC AAA GGC CGA TAC AGA
CTG TCC GGC CTG TTT GCC AAC GCT GTGCT 3'
.,,~ ,.. ... ... ... ... ... ... ... .
v GAC AGG CCG GAC AAA CGG TTG CGA C 5'
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X-612 5C-Canada _g_
b ) 5 ' CGACA ATG TTC CCA GCT ATG TCT CTA TCT GGT
-- ... .-. ... .,, ... ... ... ... ...
3 ' TGT TAC AAG GGT CGA TAC AGA GAT AGA CCA
CTG TTT GCC AAC GCT GTGCT 3 '
GAC AAA CGG TTG CGA C 5 ',
c ) 5 ' CGACC ATG GAT TTT CCG GCT ATG TCT CTG TCC
..- .~- .. .. ..- .- ... -- ~- --
3 ' TGG TAC CTA AAA GGC CGA TAC AGA GAC AGG
GGC CTG TTT GCC AAC GCT GTGCT 3 '
" ' " ' " ' ' ' ' " ' " ' '
CCG GAC AAA CGG TTG CGA C 5 ',
d) 5 ' CGATC ATG GAT TTT CCG GCT ATG TCT CTG TCC
... ..- ... ... .., -. .-- ,.. ... I.
2 0 TAG TAC CTA AAA GGC CGA TAC AGA GAC AGG
GGC CTG TTT GCC AAC GCT GTGCT 3 '
~.. ... ... ... ... ..~ .
2 5 CCG GAC AAA CGG TTG CGA C 5 ',
e ) 5 ' CGACC ATG GTT TTT CCG GCT ATG
~ " . ~ " ", , ~
3 ' TGG TAC CAA AAA GGC CGA TAC
3 0 TCT CTG TCC G~;C CTG TTT GCC
..~ ,.~ ... ... ... ... ...
AGA GAC AGG CCG GAC AAA CGG
MC GCT GTGCT 3 '
~ ... ... .
TTG CGA C 5 '
f) 5 ' CGACC ATG GCT TTT CCG GCT ATG
... .,, ... ... ... ... ...
3 ' TGG TAC CGA AAA GGC CGA TAC
TCT CTG TCC GTC CTG TTT GCC
... ... ... ... ... ... ...
AGA GAC AGG CAG GAC AAA CGG
AAC GCT GTGCT 3 '
l l l l l l l
TTG CGA C 5 '
5 0 and
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g) 5' CGATA ATG GAT TTT CCG GCT ATG
... ... ... ... ... ... ...
3' TAT TAC CTA AAA GGC CGA TAC
TCT CTG TCC GGC CTG TTT GCC
... ... ... ... ... ... ...
AGA GAC AGG CCG GAC AAA CGG
AAC GCT GTGCT 3'
... ... .
TTG CGA C 5'
wherein
A is deoxyadenyl,
G is deoxyguanyl,
C is deoxycytidyl and
T is thymidyl,
into the ~lO kb EcoRI-BamHI and ~0.6 kb BamHI-HqiAI
fragments of plasmid pCZ101 and the ~0.29 kb EcoRI-ClaI
fragment of plasmid pNM608. The resultant plasmids,
respectively designated as plasmids pCZ112, pAT1, pASPl,
pASP2, pCZ154, pCZ155 and pCZl56, contain 1) the E. coli
tryptophan gene transcriptional and translational
activating seguence (Hallewell and Emtage, 1980, Gene
9:27) in the reading frame of a coding sequence for a
bioactive bovine growth hormone derivative; 2) an
appropriately placed translational stop signal; and 3) a
runaway replicon. Cells transformed by plasmids pCZ112,
pATl, pASPl, pASP2, pCZ154, pCZ155 and pCZ156 respec-
tively express MET-ASP-ASP-LYS-bGH, MET-bGH, MET-ASP-bGH,
MET-ASP-bGH, MET-VAL-bGH, MET-ALA-PHE-PRO-ALA-MET-SER-
LEU-SER-VAL-b'GH, and MET-ASP-bGH, wherein
MET is methionine,
ASP is aspartic acid,
LEU is leucine,
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X-6125C-Canada
SER is serine,
PRO is proline,
PHE is phenylalanine,
LYS is lysine
VAL is valine,
ALA is alanine,
b'GH is the natural amino acid sequence of bovine
growth hormone beginning with amino acid 9, leucine, and
bGH is the natural amino acid sequence of bovine
growth hormone beginning with the N-terminal (first)
phenylalanine. A restriction site map of illustrative
plasmid pCZ112 is presented in Figure 10 of the accom-
panying drawings.
The plasmid pCZ101 starting material is
~10.8 kb and is constructed by ligating the ~0.6 kb
XbaI-BamHI fragment of plasmid pNM789B into similarly
digested plasmid pIM~ A3. The latter plasmid, which
contains the transcriptional and translational activat-
ing sequence of the E. coli lipoprotein gene as well as
a runaway replicon, can be obtained from E. coli K12
RV308/pIM-I'-A3, a strain deposited and made part of the
permanent stock culture collection of the Northern
Regional Research Laboratory, Peoria, Illinois. The
strain is available as a preferred source and stoc~
reservoir of the plasmid under the accession number NRRL
B-15733. The plasmid pNM789B starting material is
derived from plasmid pKEN111 in accordance with the
steps illustrated and described in Figures 1-8 and
Example 1 below. Plasmid pKEN111 can be obtained from
E. coli K12 CC620/pKEN111, a strain deposited and made
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X-6125C-Canada -12-
part of the permanent stock culture collection of the
Northern Regional Research Laboratory, Peoria, Illinois.
The strain is available as a preferred source and stock
reservoir of the plasmid under the accession number
NRRL B-15011. Plasmid pNM789B also contains the tran-
scriptional and transla~ional activating sequence of
the E. coli lipoprotein gene and, in addition, the
coding sequence, including an appropriately positioned
translational stop signal, for a fusion protein compris-
ing bGH and a nine member polypeptide at the bGH N-
terminus. Ligation of the fusion protein-coding se-
quence, contained in the XbaI-BamHI fragment, to appro-
priately cleaved plasmid pIM-I'-A3 results in the
aforementioned plasmid pCZ101 starting material.
The plasmid pNM608 starting material is
~4.6 kb and is constructed by ligating the EcoRI-ClaI
fragment of plasmid pHI7~4~1 into EcoRI-ClaI-digested
plasmid pBR322. Plasmid pHI7~4~1 can be constructed in
accordance with the teaching of Example 5 below. Be-
cause of the multiplicity of ~gI restriction sites in
plasmid pHI7~4~1, the desired EcoRI-Ta~I fragment can
best be generated by subcloning the pH17~4~1 EcoRI-HPaI
and ~ gI fragments. Plasmid pNM608 contains the E.
coli tryptophan transcriptional activating sequence and
is thus useful for constructing the present invention.
Those skilled in the art will recognize that
the various DNA linkers described above are important
components of the present invention. These sequences
can be conventionally synthesized by the modified
phosphotriester method using fully protected dideoxy-
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X-6125C-Canada -13-
ribonucleotide building blocks. Such synthetic methods
are well known in the art and can be carried out in
substantial accordance with the procedure of Itakura et
al., 1977, Science 198:1056 and Crea et al., 1978, Proc.
Nat. Acad. Sci. USA 75:5765. In addition, an especially
preferred method is disclosed in Hsiung et al., 1983,
Nucleic Acid Research 11:3227 and Narang et al., 1980,
Methods in Enæymology 68:90. The linkers code for a
translational activating sequence and also amino acids
comprising the first portion (~-terminus region) of the
aforementioned bGH derivative compounds. The remainder
of the bGH coding sequence (including an appropriately
positioned translational stop signal) and also a tran-
scriptional activating sequence can be provided by
ligation of the appropriate fragment of plasmid pCZ101.
Such ligations result in the illustrative bovine growth
hormone derivative expression plasmids of the present
invention.
Deletion of the ~900 bp BstEII restriction
fragment of plasmid pCZ101, followed by recirculariza-
tion, results in plasmid pCZ103. The BstEII deletion
does not affect the bGH coding region in plasmid pCZ101.
Using plasmid pCZ103 in place of plasmid pCZ101 in the
above-described constructions thus results in similar,
but 900 bp smaller, plasmids. The bGH derivatives ex-
pressed by these pCZ103-derived plasmids are therefore
identical to the derivatives expressed by their pCZ101-
derived counterparts.
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X-6125C-Canada -14-
Thus, using the ~9.3 kb BamHI-XbaI and ~G.6 kb
BamHI-HqiA1 restriction fragments of plasmid pCZ103, in
place of the ~10.2 kb BamHI-XbaI and 0.6 kb BamHI-HgiAI
restriction fragments of plasmid pCZ101, in the above-
described constructions of plasmids pJRl and pAT2 re-
sults in the construction of derivative plasmids pJRl.3
and pAT2.3, respectively. Using the ~9.3 kb BamHI-EcoRI
and ~0.6 kb BamHI-HqiAl restriction fragments of plasmid
pCZ103, in place of the ~10.2 kb BamHI-EcoRI and ~0.6 kb
BamHI-HqiA1 restriction fragments of plasmid pCZ101, in
the above-described constructions of plasmids pATl,
pASP1, pASP2, pCZ154, pCZ155, and pCZ156 results in the
construction of derivative plasmids pAT1.3, pASP2.3,
pCZ154.3, pCZ155.3, and pCZ156.3, respectively.
The present invention is in no way limited to
the use of a particular transcriptional activating
sequence since the choice of a specific sequence is not
critical to the operability of the present invention.
Transcriptional activating sequences which can be sub-
stituted for the previously exemplified lipoprotein and
tryptophan activating sequences include, but are not
limited to, the _. coli lactose (lac), bacteriophage
A PLOL and bacteriophage A PROR transcriptional activat-
ing sequences. In addition, one or more transcriptional
2S activatinq sequences, or parts of them, can be used in
tandem, such as, for example, the trP and lac or tac
transcriptional activating sequences. All of the
aforementioned sequences have been previously charac-
terized and can be constructed either synthetically or
from known plasmids.
X-6125C-Canada -15-
In the specific embodimPnts herein described,
plasmid replication is determined by a thermoinducible
runaway replicon disclosed in both GB Patent Publication
Number 1,557,774 and Uhlin et al., 1979, &ene 6:91. At
temperatures below 30C, especially 25C, the replicon
maintains a relatively low copy number of about 10-15
copies per cell. When the temperature is raised to
37C, copy number control is lost and plasmids contain-
ing the replicon amplify to 1000-2000 copies per cell.
The particular runaway replicon exemplified herein is
contained in the previously described plasmid pIM~ A3
starting material. Skilled artisans will understand
that the present invention is not limited to the use of
any particular runaway replicon or copy number mutant.
Other inducible runaway or high copy number replicons
can be obtained by appropriate selection or can be
constructed in accordance with the procedure disclosed
in International Publication Number WO82/02901. Such
replicons can be used to construct expression vectors
that are also within the scope of the present invention.
The cloning of foreign genes into vectors
containing a runaway replicon results, upon induction
and loss of copy number control, in a greatly increased
rate of protein synthesis and the concomitant formation
of a heretofore unknown and unrecognized species of
intracellular proteinaceous granule. Such granules,
which further comprise the present invention, are highly
homogeneous,in their protein composition and are thus
distinguishable over the known high molecular weight
aggregates and inclusions that sometimes occur in
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X-6125C-Canada -16-
recombinant DNA-containing host cells. The latter
inclusions are heterogeneous, commonly containing an
assortment of cellular components, such as nucleic
acids, carbohydrates, lipids and peptides, and comprise
only 10-20% of a particular protein product. The gran-
ules of the present invention are highly homogeneous
with the desired protein product comprising at least
50% and often exceeding 80% of the granule.
The present novel species of granule can be
readily isolated from cell lysates and is stable to
washing in low concentrations of urea or detergents.
Washing removes proteins that bind non-specifically to
the granule. Isolation, which generates high specific
activity material, constitutes a useful first step in
the purification of foreign proteins. All subsequent
steps in purification are therefore simpliied. Par-
ticularly helpful is the fact that cell wall components
can be readily separated from the granules.
It is believed that the formation of the
granules of the present invention seguesters a foreign
protein such that disruption of normal cell metabolism
and premature cell death does not occur. Some proteins,
such as human proinsulin, are rapidly degraded in E.
coli and do not accumulate. However, when such proteins
are coded for in vectors containing a runaway replicon,
the proinsulin protein forms insoluble granules that
protect the ordinarily unstable protein from proteolytic
degradation. The formation of the present species of
granule is thus useful not only for accumulating unsta-
ble proteins, but also for simplifying procedures forisolating and purifying gene products.
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X-6125C-Canada -17-
The present invention, in another aspect, therefore,
further comprises a method for producing an intracellular highly
homogeneous species of foreign proteinaceous granule, said
method comprising
1) transforming a competent cell with a
selectable and autonomously replicating
recombinant DNA expression vector which
comprlses
a) a runaway replicon, and
b) a transcriptional and translational
activating sequence which is in the
re~ding frame of a nucleotide
sequence that codes for a functional
polypeptide, said functional poly-
peptide coding sequence containing a
translational stop signal positioned
immediately adjacent and downstream
from the nucleotide triplet that
codes for the carboxy terminus of
said functional polypeptide, and
2) culturing the transformed cell under
growth conditions suitable for gene
expression and activation or induction of
said runaway replicon.
Any runaway replicon and transcriptional and
translational activating sequence, as previously de-
scribed herein, and virtually any nucleotide sequence
that codes for a functional polypeptide or fused gene
produc~ can be used to construct vectors for use in the
above-defined method. Such coding sequences include,
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X-6125C-Canada -18-
but are not limited to, sequences that code for bovine
growth hormone (bGH), human growth hormone (hGH), human
pre-growth hormone (pre-hGH), porcine growth hormone
(pGH), mammalian growth hormone, avian growth hormone,
growth hormone releasing factor, human insulin A chain,
human insulin B chain, hum~n proinsulin, human pre-
proinsulin, human and non-human interferon, urokinase,
tissue plasminogen activator, interleukin II, any
polypeptide hormone, any polypeptide enzyme and any
bioactive polypeptide of research or commercial value.
Transformed cells which, upon culturing in
accordance with the above-defined method, exemplify the
production of the present species of granule include,
but are not limited to, E. coli K12 RV308/pCZl920, E.
coli K12 ~V308/pJRl, E. coli K12 RV308/pASPl, E. coli
K12 RV308/pCZ112, E, coli K12 RV308/pASP2, E. coli K12
RV308/pAT1, E. coli K12 RV308/pAT2, E. coli K12
RV308/pCZ154, E. coli K12 RV308/pCZ156, E. coli K12
RV308/pJR1.3, E. coli K12 RV308/pASPl.3, E. coli K12
RV308/pASP2.3, E. coli K12 RV308/pATl.3, E. coli K12
RV308/pAT2.3, E. coli K12 RV308/pCZl54.3, and E. coli
K12 RV308/pCZ156.3.
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X-6125C-Canada -19-
The present method for producing a highly
homogeneous species of granule is not limited to the use
of the above plasmids coding for bovine or human growth
hormone. Human proinsulin granules can also be produced
by culturing cells, in accordance with the aforemen-
tioned method, that contain vectors comprising the human
proinsulin coding sequence. Such a vector can be con-
structed by ligating a trPLE'-proinsulin fusion
sequence-containing EcoRI-BamH~ fragment, as depicted
in Figure 15, into the large EcoRI-BamHI fragment of
plasmid pCZ101. The resultant plasmid, designated as
plasmid pCZ201, can transform E. coli, such as E. coli
K12 RV308, in accordance with conventional transforma-
tion methods. The present granules, which in this case
comprise human proinsulin, can then be produced by
culturing the E. coli K12 RV308/pCZ201 transformants
at 37C.
Many modifications and variations of the
present illustrative DNA sequences and plasmids are
possible. For example, the degeneracy of the genetic
code allows for the substitution of nucleotides through-
out polypeptide coding regions as well as for the sub-
stitution of the TAG or TGA translational stop signals
.... ...
ACT ACT
for the TAA translational stop signal specifically
. . .
ATT
exemplified. Thus R, as defined hereinabove, can be one
of any number of possible DNA sequences that code for
amino acids 9 (leucine) through 191 (phenylalanine) of
~ ~9~7~3
X-6125C-Canada
bGH. Such sequences can be deduced from the known amino
acid sequence of bGH and can be constructed by following
conventional synthetic procedures. However, nucleotide
triplets should be chosen in accordance with known
principles and extrapolations reviewed in Zuker and
Stiegler, 1981, Nucleic Acids Research 9(1):133, to
avoid generating complementary bases in mRNA. Hydrogen
bonding (pairing) between such complementary bases
results in stem and loop configurations and folding that
reduce the efficiency of translation. Skilled artisans
will understand that all of the modifications and vari-
ations disclosed above can be conven~ionally synthesized
in substantial accordance with the synthetic methods
previously cited. Therefore, the present invention is
in no way limited to the DNA sequences and plasmids
specifically exemplified.
The expression vectors and method of this
invention can be applied to a wide range of host organ-
isms, for example, gram-negative prokaryotic organisms
such as Escherichia coli, E. coli K12, E. coli Kl2
RV308, E. coli K12 HBlOl, E. coli K12 C600, E. coli K12
C600 Rk-Mk-, E. coli K12 RR1, E. coli K12 MM294 and the
like. Although all of the embodiments of the present
invention are useful, some of the vectors and trans-
formants are preferred. Preferred vectors includepCZ1920, pJRl, pJRl.3, pCZ112, pATl, pATl.3, pAT2,
pAT2.3, pASP1, pASPl.3, pCZ154, pCZ154.3, pCZl56,
pCZ156.3, pASP2.3, and pASP2 and preferred transform-
ants include E. coli K12 RV308/pCZ1920, E. coli K12
RV308/pJRl, E. coli K12 RV308/pJRl.3, E. coli K12
~s9~8
X-6125C-Ca~ada -21-
RV308/pCZ112, E. coli K12 RV308/pAT1, E. coli K12 RV308/
pAT1.3, E. coli K12 RV308/pAT2, E. _li K12 RV308/p~T2.3,
E. coli K12 RV308/pASPl, E. coli K12 RV308/pASPl.3,
E. coli K12 RV308/pCZ154, E. coli K12 RV308/pCZ154.3,
E coli K12 RV308/pCZ156, E. coli K12 RV308/pCZ156.3,
E. coli K12 RV308/pASP2.3, and E. coli K12 RV308/pASP2.
Of this preferred group, plasmids pCZ112, pCZ154,
pCZ154.3, pCZ156, pCZ156.3, pASP2.3, and pASP2 and
transformants E. coli K12 RV308/pCZ112, E. coli K12
RV308/pCZ154, E. coli K12 RV308/pCZ154.3, E. coli K12
RV308/pCZ156, E. coli K12 RV308/pCZ156.3, E. coli K12
RV308/p~SP2.3, and E. coli K12 RV398/pASP2 are espe-
cially preferred.
Thoce skilled in the art will recognize that
1~ the expre~sion vcctors of this invention are used to
transform ~uitable host organi~ms ~uch that a bovine
growth hormone derivative product i8 expressed using
standard fermentation conditions. The product, ex-
pressed as a highly homoge~eous granule and isolated
by routine methods from the resulting cell lysates is
useful for increasing milk production and for gener-
ally stimulating growth in cattle. Modes of use,
admini6tration and suitable do6age~ for cattle are known
and disclosed in pages 7 - 9 of European Patent Application
No. 0 085 036 of Monsanto Company, published August 3, 19~3.
The following examples further illustrate the invention
disclosed herein. Both an explanation of and the actual
procedures for constructing the invention are described
where appropriate.
. (
~9~718
X-612 5C-Canada -22 -
Exam~le 1
Construction of Plasmid ~NM789B
A. Construction of Plasmid pKEN021 and the XbaI-BamHI
Fraqment Thereof
The ~5.1 ~b fragment produced by XbaI-BamHI
cleavage of plasmid pKEN021 (106 in Figure 3) was used
as starting material. Plasmid pKEN021 is a derivative
of pKENlll, (101 in Figure 1 and further described in
Lee, et al., 1981, J. Bact. 146: 861-866 and Zwiebel, et
_., 1981, J. Bact. 145: 654-656), which is on deposit
in E. coli CC620 (NRRL Deposit No. 15011) and which has
a ~2.8k~ fragment which contains the lipoprotein gene of
E. coli. A description of this fragment is provided in
Naka~ura and Inouye, 1979, Cell 18: 1109-1117. In
pXEN021, the 650 bp (base pair) sequence between the
unique EcoRI and SalI restriction sites of pBR322 has
been replaced by sequences taken from the lipoprotein
gene of E. coli. The lipoprotein gene sequence (Nakamura
and Inouye, 1979) includes a 462 bp AluI fragment,
upstream from the first triplet (methionine) of the
lipoprotein gene that contains the promoter, the 5'
untranslated region and the ribosome binding site. A
unique XbaI restriction site is located within the
ribosome binding site 16 bp before the translation
initiating methionine signal. A PvuII restriction site
located 105 bp upstream from the translation termination
codon of the structural gene was,changed to a BamHI
~91~
X-6125C-C~nada -23-
restriction site by the addition of a synthetic DNA
linker (S' CCGGATCCGG3 ', obtained from Collaborative
Research). The coding sequence for the last thirty-five
amino acids of lipoprotein, the translati~n termination
signal, and the sequence corresponding to the 3' un-
translated region of the messenger RNA follow the BamHI
site. Plasmid pKEN021 also includes some 850 bp of
extraneous sequences unrelated to the lipoprotein gene
and located downstream of it in the E. coli chromosome.
These sequences were included as a consequence of the
methods and restriction enzyme sites used in the origi-
nal isolation of the gene.
Referring to Figures 1, 2, and 3, plasmid
pKEN021 is derived from pKENlll in the following manner:
~bout 50 ~g of pKENlll (101 in Figure 1) are digested
with 25 units of ~p~II restriction enzyme in 300 ~1 of a
buffer containing 20mM Tris HCl, pH 7.4, lOmM MgC12, and
6mM ~-mercaptoethanol at 37C for 2 hours. The mixture
is extracted twice with 300 ~1 of a 50:50 mixture of
phenol and chloroform and the recovered aqueous phase is
then precipitated with 2.5 volumes of ethanol and 0.1
volumes of 3M sodium acetate. The DNA pellet is dis-
solved in 100 ~1 of electrophoresis buffer and fraction-
ated on a 5 percent polyacrylamide gel (acrylamide:bis
ratio is 29:1 in all gels except where noted). The gel
is stained in a solution containing 0.5 ~g/ml of ethidium
bromide and bands are visualized under long wave-length
ultraviolet light. A 950 bp band is isolated and
recovered from the gel by electroelution into a dialysis
bag. After phenol/CHC13 extraction and ethanol precipi-
1~9~7~8
X-6 12 5C-Canada -24-
tation, the recovered DNA (approximately 2.5 ~g~ is
dissolved in 25 ~l of TEN (lOmM NaCl, lOmM Tris HCl
pH 7.4 and lmM sodium ethylenedir.itrilotetraacetate
(EDTA~, pH 8.0).
S About 2 ~g of the 950 bp HpaII fragment are
digested with AluI restriction enzyme in 200 ~l of a
buffer containing 50mM NaCl, 6mM Tris-HCl (pH 7.6), 6mM
MgCl2, and 6mM ~-mercaptoethanol for 2 hours at 37C.
The DNA is fractionated on a 6 percent polyacrylamide
gel and the 462 bp AluI fragment generated is recovered
`and purified by the method previously described. The
462 bp AluI fragment (approximately l ~g) is dissolved
in lO ~l of T4 DNA ligase buffer (66mM Tris-HCl pH 7.6,
lOmM MgC12, lOmM dithiothreitol, O.4mM ATP) containing
150 picomoles of phosphorylated EcoRI linker (5'GGAATTCC3'
from Collaborative Research) and 2 units T4 DNA ligase.
After incubation at 4C. for 16 hours, the mixture is
heated at 65C. for lO minutes and diluted to 100 ~l
with the addition of EcoRI buffer (lOOmM Tris-HCl pH 7.2,
50mM NaCl, lOmM MgC12, 6mM ~-mercaptoethanol) and 40
units EcoRI enzyme. After 2 hours at 37C., the sample
is conventionally phenol/CHC13 extracted and ethanol
precipitated. The DNA is then dissolved in 20 ~1 of T4
DNA ligase buffer containing 0.1 unit T4 DNA ligase and
0.1 ~g pBR322 (102 in Figure l) which has been linearized
with E RI and then treated with alkaline phosphatase.
After ligation at 4C for 16 hours, the resultant DNA is
used to conventionally transform E. coli strain K12
HB101. Transformants are selected on agar plates
containing 12 ~g/ml of tetracycline and plasmids isolat-
1;~91~
X-6125C-Canada -25-
ed from resistant colonies by the rapid alkaline extrac-
tion procedure described in Birnboim and Doly, 1979,
Nucleic Acids Research 7: 1513-1523. A plasmid (103 in
Figure 1) containing a 466 bp XbaI-BamHI fragment is
selected and used as the starting material for the step
next described.
About two ~g of this plasmid (103 in Figure 2)
are digested with 2 units of HlndIII enzyme in 50 ~1
HlndIII buffer (60mM NaCl, lOmM Tris HCl, pH 7.4, lOmM
MgC12 and 6mM ~-mercaptoethanol) for 1 hour at 37C.
After phenol/CHC13 extraction and ethanol precipitation,
the DNA is dissolved in 200 ~1 of a buffer containing
300mM NaCl, 30mM sodium acetate pH 4.25, lmM ZnC12 and
200 units of Sl nuclease (Miles Laboratories). After 1
hour at 15C., the reaction is stopped by phenol/CHC13
extraction and ethanol precipitation. The resultant DNA
is dissolved in 10 ~1 T4 DNA ligase buffer containing 20
picomoles phosphorylated BamHI linkers (S'CCGGATCCGG3',
from Collaborative Research) and 2 units T4 DNA ligase.
After 16 hours at 4C, the reaction mixture is heated at
65C for 10 minutes to inactivate the ligase and then
diluted to 100 ~1 in B mHI buffer (150mM NaCl, 20mM
Tris HCl, pH 8.0, lOmM MgC12, 6mM ~-mercaptoethanol)
containing 20 units BamHI enzyme. After 2 hours at
37C, the mixture is purified on a 1 percent agarose
gel. The gel is stained and the larger fragment
(~4.5 kb) is recovered by elution after freezing and
then purified by pheno}/CHC13 extraction and ethanol
precipitation. The recovered fragment with BamHI
cohesiv~ ends is dissolved in 20 ~1 of T4 DNA ligase
917~8
X-6125C-Canada -26-
buffer containing 0.1 unit T4 DNA ligase. After 16hours at 4C, the DNA is used to transform E. coli
HB101. Transformants are selected by resistance to
ampicillin (Apr) at 100 ~g/ml and screened for sensi-
tivity to 10 ~g/ml tetracycline (Tcs). Several
plasmids, prepared by the previously described Birnboim
procedure from colonies which are AprTcs, are examined
for the absence of a HindIII site and the presence of a
single BamHI site. EcoRI, SalI sequential digestion
yields a 466 bp and a 305 bp fragment. A plasmid (104
in Figure 2) with these characteristics is selected and
then modified to convert the EcoRI site, located up-
stream of the 1PP promoter, to a H1ndIII restriction
site.
Two micrograms of plasmid (104 in Figure 2)
are digested in 100 ~1 of EcoRI buffer with 0.2 units of
EcoRI for 10 minute3 at 37C. The reaction is stopped
by heating for 10 minutes at 65C and then, after
phenol/CHC13 extraction, the DNA is ethanol precipitated,
dissolved in 200 ~1 of S1 nuclease buffer containing Sl
nuclease at 1000 units/ml and reacted at 12C for 1
hour. The reaction is stopped by phenol/CHC13 extrac-
tion and ethanol precipitation. The resultant DNA is
resuspended in 10 ~1 of T4 DNA ligase buffer containing
20 picomoles phosphorylated HindIII linker (5'CCAAGCTTGG3',
from Collaborative Research) and 2 units of T4 DNA
ligase. After 16 hours at 4C, the mixture is heated
for 10 minutes at 65C, diluted to 150 ~1 in HindIII
buffer containing 10 units HindIII enzyme, incubated for
2 hours at 37C and then fractionated on a 1 percent
~2~9~'7~8
X-6125C-Canada -27-
agarose gel. The largest band (equivalent to single cut
plasmid) is conventionally recovered and purified,
dissolved in 20 ~1 T4 ligase buffer containing 0.2 units
T4 ligase, incubated 16 hours at 4C. and then used to
transform E. coli B101. Transformants are selected for
ampicillin resistance and plasmid isolates conventional-
ly analyzed by restriction enzyme analysis. A plasmid
(105 in Figure 2) with an EcoRI-HindIII fragment of
500 bp is selected and used as the cloning vector for
addition of the 3' region of the 1PP gene.
About two ~g of plasmid (105 in Figure 3) are
digested in 50 ~1 of SalI restriction buffer (150mM
NaCl, 6mM Tris HCl, pH 7.9, 6mM MgC12, 6mM ~-mercapto-
ethanol) with 2 units of SalI for 1 hour at 37C and
then diluted to 150 ~1 in BamHI buffer containing 2
units BamHI. After 1 hour at 37C, 2.5 units of alka-
line phosphatase are added and then incubation is
continued or 1 hour at 65C. The material is
phenol/CHC13 extracted, ethanol precipitated, dissolved
in TEN, and used as a cloning vector for the 1PP 3'
fragment.
To obtain the fragment containing the 1PP 3'
region, 10 ~g of pKENlll (101 in Figure 3) are digested
in 200 ~1 of HPaI buffer (20mM KCl, lOmM Tris HCl, pH
7.4, lOmM MgC12 and 6mM ~-mercaptoethanol) with 10 units
of HpaI for 2 hours at 37~C. After phenol/CHC13 extrac-
tion and ethanol precipitation, the DNA is dissolved in
10 ~1 T4 DNA ligase buffer containing 20 picomoles
phosphorylated SalI linker ~5'GGTCGACC3', from Collabo-
rative Research) and 2 units T4 DNA ligase and then
~91
X-6125C-Canada -28-
incubated for 16 hours at 4C. The ligase is inacti-
vated by heating at 65C for 10 minutes. The resultant
material is diluted to 100 ~1 in SalI buffer containing
10 units of SalI and incubated 1 hour at 37C, and then
diluted to 300 ~1 in PvuII buffer (60mM NaCl, 6mM
Tris-HCl, pH 7.5, 6mM MgC12, 6mM ~-mercaptoethanol)
containing 10 units PvuII restriction en~yme. After 1
hour at 37C, the DNA is fractionated on a 5 percent
polyacrylamide gel. Approximately 0.5 ~g of a 950 bp
fragment is recovered, purified and dissolved in TEN.
Two-tenths microgram of the fragment is diluted into
20 ~1 T4 DNA ligase buffer containing 20 picomoles
phosphorylated BamHI linker (5'CCGGATCCGG3', from
Collaborative Research) and 2 units T4 DNA ligase and
then incubated for 16 hours at 4C. The resultant DNA
is then heated for 10 minutes at 65C, diluted to 100 ~1
in BamHI buffer containing 20 units BamHI, incubated at
37C or 2 hours and then fractionated on a 5 percent
polyacrylamide gel to remove excess linker molecules.
The resultant 950 bp fragment having BamHI and SalI
cohesive ends is conventionally purified and dissolved
in 20 ~1 of T4 DNA ligase buffer containing both 0.2 ~g
of the cloning vector described previously and 0.2 units
T4 DNA ligase. After incubation for 16 hours at 4C,
the DNA is used to transform E. coli K12 B 101. Plasmids
are prepared from ampicillin resistant transformants and
conventionally analyzed for the SalI-BamHI fragment.
The desired plasmid (~5.2 kb) is designated pKEN021 (106
in Figure 3).
~91~7~8
X-6125C-Canada -29-
Ten micrograms of pKEN021 were digested at
37C in 200 ~1 of XbaI/BamHI buffer (150mM NaCl, lOmM
Tris-HCl, pH 8, lOmM MgC12, 6mM ~-mercaptoethanol) using
10 units of BamHI for 1 hour followed by lO units of
XbaI for an additional hour at 37C. The desired XbaI-
BamHI-digested DNA was then treated with 2.5 units of
alkaline phosphatase for 1.5 hours at 65C, phenol/CHC13
extracted, collected by ethanol precipitation, and
dissolved in 50 ~1 of TEN for future use (107 in
Figure 3).
B. Construction of Plasmid pNM575
Plasmid ptrpED50chGH800 (108 in Figur~ 4),
described in Martial et al., 1979, Science 205: 602-607
was used as the source of a DNA fragment containing the
coding sequence for a portion of the human growth
hormone gene. This fragment can also be constructed
synthetically (Itakura et al., 1977 and Crea et al.,
1978) or can be obtained using recognized methodology
described by Goodman et al., 197g, Methods in Enzymology
68:75-90, by isolating mRNA coding for human growth
hormone from human pituitaries. The human growth
hormone gene portion of plasmid ptrpED50chGH800 contains
a unique SmaI restriction site 6 bp downstream from the
translation termination codon of the gene. This site
was changed to a BamHI site using the following proce-
dure: 6 ~g of the plasmid were digested with 6 units of
SmaI in 200 ~1 of SmaI restriction buffer (15mM Tris-HCl,
pH 8.0, 6mM MgC12, 15mM KCl and 6mM ~-mercaptoethanol)
~9~7~8
X-6125C-Canada -30-
for 1.5 hours at 37C. After digestion was complete,
phenol/CHC13 extraction was performed and the DNA was
recovered by ethanol precipitation and then dissolved in
24 ~1 of TEN. Forty picomoles of phosphorylated BamHI
adapter fragment (Collaborative Research) were added to
0.5 ~g (0.2 picomole ends) of the above-digested plasmid
in 16 ~1 of ligase buffer containing 2 units T4 DNA
ligase. The mixture was incubated 2 hours at 22C, 16
hours at 4C and then 10 minutes at 6SC. BamHI cohe-
sive termini were generated by conventional digestionwith BamHI restriction enzyme. The enzyme cleaved the
linker sequence as well as the BamHI site located at the
beginning of the cloned human growth hormone cDNA
sequence. This yielded a 691 bp fragment with cohesive
BamHI ends which was separated on a 6 percent polyacryl-
amide gel and then conventionally recovered. The
recovered DNA fragment was ligated (using 0.2 unit T4
DNA ligase in 20 ~1 of buffer under previously described
conditions) with 0.2 ~g of BamHI-digested and alkaline
phosphatase-treated pBR322 (102 in Figure 4). After 16
hours at 4C, the material was used to transform E. coli
strain JA221 (NRRL No. B-15014) in substantial accor-
dance with the transformation procedure of Wensink et
al., 1974, Cell 3:315-325. Transformants were selected
on agar plates containing 100 ~g/ml ampicillin and then
plasmids were conventionally isolated and identified by
restriction enzyme and gel electrophoretic analysis.
Desired plasmids, designated as pNM575 (109 in Figure 4),
contain a BamHI fragment of approximately 700 bp and
were conventionally amplified for future use.
X-6125C-Canada -31-
C. Construction of Plasmid ~NM702
The DNA sequence of mature human growth
hormone contains one FnuDII site which is 47 bp from the
S first nucleotide. Twenty-five micrograms of pNM575 were
digested in 250 ~1 of BamHI buffer with 25 units of
BamHI at 37C for 1 hour. The 691 bp fragment with
BamHI cohesive termini was conventionally lsolated from
a 6 percent polyacrylamide gel and purified. After
purification of the fragment, one third of i~ (equiva-
lent to 8 ~g of plasmid) was digested in 100 ~1 of
FnuDII buffer (6mM NaCl, 6mM Tris HCl, pH 7.4, 6mM
MgC12, 6mM ~-mercaptoethanol) with 2.5 uni~s FnuDII for
1.5 hours at 37C. Electrophoresis on a 6 percent
polyacrylamide gel and standard recovery procedures were
used to isolate a 538 bp DNA fragment containing the
coding sequence for the last 175 amino acids of the gene
followed by a translation stop signal.
A double stranded DNA fragment (110 in Figure 5)
was synthesized by the phosphotriester method to join
the 1PP promoter region with the human growth hormone
coding region. The double stranded DNA fragment (110 in
Figure 5) has the following seguence:
XbaI
5' CTAGAGGGTATTAATAATGTTCCCATTGGATGATGATGATAAGTTCCCAA-
......... -.. -
3' TCCCATAATTATTACAAGGGTAACCTACTACTACTATTCAAGGGTT-
CCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCG 3- FnuDII
......................... --.. -
GGTAAGGGAATAGGTCCGAAAAACTGTTGCGATACGAGGC 5'
~'~9~7~8
X-6125C-Canada -32-
The fragment was prepared by recognized
phosphotriester methodology by which the following
segments were prepared:
1) CTAGAG>AT
2) TAATAATGTTCC
3) CATTGGATGAT
4) GATGATAAGTTCC
5) CAACCATTCCC
6) TTATCCAGGC
7) TTTTTGACAACG
8) CTATGCTCCG
9) CATTATTAATACCCT
10) ATGGGAA
11) CTTATCATCATCATCCA
12) GGTTGGGAA
13) GGATAAGGGAAT
14) GTCAAAAAGCCT
15) CGGAGCATAGCGTT
Using the above-prepared segments, the T4
ligase catalyzed joining reactions were performed
stepwise as described below:
a) 5'-Unphosphorylated segment 1 was joined to
5'-phosphorylated segment 2 in the presence of
5'-phosphorylated segment 9 using T4 ligase to form DNA
duplex 1 (Brown et al., 1979, Methods in Enzymology
68;109-151). The duplex was isolated by preparative gel
electrophoresis on 15% polyacrylamide.
~L~917~
X-6125C-Canada -33-
b) S'-Phosphorylated segment 3 was joined to
5'-phosphorylated segment 4 in the presence of
5'-phosphorylated segment 11 using T4 ligase to form DNA
duplex 2 which was purified by 15% polyacrylamide gel
electrophoresis.
c) 5'-Phosphorylated segment 5 was joined to
5'-phosphorylated segment 6 in the presence of 5'-
phosphorylated segments 12 and 13 using T4 ligase to
form DNA duplex 3 which was purified by lS~ polyacryl-
0 amide gel electrophoresis.d) 5'-Phosphorylated segment 7 was joined to
5'-phosphorylated segment 8 in the presence of 5'-
phosphorylated segment 14 and 5'-unphosphorylated
segment 15 using T4 ligase to form DNA duplex 4 which5 was purified by 15% polyacrylamide gel electrophoresis.
e) The DNA duplexes 2, 3 and 4 then were
joined together by T4 ligase to form DNA duplex 5 which
was purified by 15% polyacrylamide gel electrophoresis.
f) 5'-phosphorylated segment 10 and DNA
duplex 5 were added, in the presence of T4 ligase, to
the DNA duplex 1. The resulting DNA duplex (110 in
Figure 5) was purified by 10% polyacrylamide gel
electrophoresis. This DNA duplex then was enzymatically
phosphorylated using T4 polynucleotide kinase and
[y-32P]ATP by following established procedure.
The expression plasmid pNM702 was constructed
by enzymatically joining 0.1 picomole (0.4 ~g) of the
XbaI-BamHI fragment of plasmid pKEN021 ~107 in Figure 5),
0.025 picomoles synthetic DNA fragment (110 in Figure 5)
and 0.3 picomoles ~0.08 ~g) of 538 bp fragment ~from 109
l?t91718
X-6125C-Canada _34_
in Figure ~) in 24 ~1 of ligation buffer using 1.5 units
T4 DNA ligase. After incubation for 16 hours at 4C,
the mixture was used to transform E. coli JA221 as
previously described. Transformants were selected on
agar plates containing 100 ~g/ml ampicillin and were
conventionally cultured as a preferred source of the
desired expression plasmid.
Expression of human growth hormone was detect-
ed by a standard radioimmunoassay procedure (Twomey et
al., 1974, Clin. Chem. 20:389-391) and was determined to
be at least 2 million molecules per cell.
D. Construction of Plasmid PNM789
Plasmid pNM702 (111 in Figure 6), the expres-
sion plasmid for human growth hormone, was used as the
starting material for construction of a pla~mid express-
ing Met-Phe-Pro-Leu-Asp-Asp-Asp-Asp-Lys-bGH.
Plasmid pBP348 (112 in Figure 6), described in
Miller et al., 1980, J. Biol. Chem. 255:7521-7524, was
used as the source of two DNA fragments containing the
coding sequence for a portion of the bovine growth
hormone gene. The plasmid contains an 831 bp bovine
growth hormone-encoding sequence cloned in the PstI
restriction site of pBR322. As an alternative to the
method described in Miller et al., 1980, the seguence
for bovine growth hormone can be constructed syntheti-
cally (Itakura et al., 1977 and Crea et al., 1978) or
can also be obtained from messenger RNA isolated from
bovine pituitaries by the now routine procedures de-
scribed by Goodman et al., 1979.
~X9~7~
X-6125C-Canada _35_
The coding sequences for human growth hormone
and bovine growth hormone are very similar and show much
homology. Particularly useful in the construction of
the expression plasmid for bovine growth ~ormone were
S the fragments generated by digestion with the restric-
tion enzyme P II. The size of the fragments produced
are 497 bp in human growth hormone and 494 bp in bovine
growth hormone and the corresponding restriction sites
occur in the same reading frames in both sequences.
Ten micrograms of pNM702 (111 in Figure 6)
were partially digested with 1 unit of PvuII in 200 ~1
of PvuII restriction buffer (60mM NaCl, 6mM Tris HCl, pH
7.5, 6mM MgC12, 6mM ~-mercaptoethanol) for 10 minutes at
37C. After the reaction was stopped by heating at 65C
for 10 minutes, the DNA was treated with alkaline
phosphatase and the fragments separated on a one percent
agarose,gel. The linear DNA fragment ~113 in Figure 6)
of the size that corresponded to DNA with the 497 bp
Pw II fragment missing (runs slightly faster than single
cut plasmid) was conventionally excised, purified and
used in the construction of an intermediate plasmid (114
in Figure 6).
A 494 bp PvuII fragment of plasmid pBP348 was
prepared by digesting 10 ~g of the plasmid in 200 ~1
PvuII buffer containing 10 units of PvuII for 1 hour at
37C. The fragments were separated on a 6 percent
polyacrylamide gel and the desired 494 bp fragment (from
112 in Figure 6) was conventionally visualized and
purified.
P?~91~7~8
X-6125C-Canada -36-
Intermediate plasmid (114 in Figure 6) was
constructed by reacting 0.2 ~g of the plasmid pNM702
PvuII fragment with 0.05 ~g of 494 bp fragment in 20 ~1
of T4 DNA ligase buffer containing 2 units T4 DNA ligase
for 16 hours at 4C. After transformation and selection
of transformants for ampicillin resistance, the plasmids
were conventionally analyzed for the presence and proper
orientation of the 494 bp PvuII fragment. Plasmids with
a 494 bp PvuII fragment and a 440 bp XbaI-SmaI fragment
are selected for use in further constructions.
Ten micrograms of the intermediate plasmid
(114 in Figure 7) were digested with 1 unit PvuII in
200 ~1 PvuII buffer for 5 minutes at 37C. After the
heating at 65C for 10 minutes, the mixture was spread
on a 1 percent agarose gel and linear DNA having only a
single PvuII cut per molecule was recovered and puri-
fied. This recovered material (approximately 3 ~g) was
digested completely with 5 units of XbaI and treated
with alkaline phosphatase. The fragments were spread on
a 1 percent agarose gel and the largest fragment (miss-
ing the 109 bp fragment between the Xbal and the first
PvuII site in human and bovine growth hormone) was
conventionally recovered (115 in Figure 7).
The DNA sequence for the first 23 amino acids
(69 bp) of bovine growth hormone to the first PvuII site
contains 2 HPaII restriction sites, the first of which
is 23 bp from the first nucleotide of the coding se-
quence. A 63 bp fragment (116 in Figure 7) was synthe-
sized by the phosphotriester method, This fragment
corresponds to the 19 bp sequence from the XbaI site in
1?~917~ 8
X-6125C-Canada 37
the 1PP ribosome binding site through the ATG transla-
tional start signal followed by the coding sequence for
Phe-Pro-Leu-Asp-Asp-Asp-Asp-Lys (24 bp) and 20 nucleo-
tides of the coding sequence of bovine growth hormone
S (from Phe to the first H~aII site). The fragment has
the following sequence:
XbaI
5' CTAGAGGGTATTAATAATGTTCCCATTGGATGATGATGATAAG-
.............................. -I------
3' TCCCATAATTATTACAAGGGTAACCTACTACTACTATTC-
TTCCCAGCCATGTCCTTGTC 3, HPaII
....................
AAGGGTCGGTACAGGAACAGGC 5'
In producing the 63 bp fragment, the following
nine segments were prepared:
1) CTAGAGGGTAT
2) TAATAATGTTCC
3) CATTGGATGAT
4) GATGATAAGTTCC
5) CAGCCATGTCCTTGTC
6) ATGGGAACATTATTAATACCCT
7) TTATCATCATCATCCA
8) ATGGCTGGGAAC
9) CGGACAAGGAC
Using the above-prepared segments, the T4
ligase catalyzed joining reactions were performed
stepwise as described below:
a~ 5'-Unphosphorylated segment 1 was joined to
5'-phosphorylated segment 2 in the presence of 5'-
~91~
X-6125C-Canada -38-
phosphorylated segment 6 using T4 ligase to form DNA
duplex 1 which was purified by 15% polyacrylamide gel
electrophoresis.
b) 5'-Phosphorylated segments 3, 4 and 5 were
joined in the presence of 5'-phosphorylated segments 7
and 8 and 5'-unphosphorylated segment 9 using T4 ligase
to form DNA duplex 2 which was purified by 15% poly-
acrylamide gel electrophoresis.
c) Duplexes 1 and 2 then were joined by T4
ligase to form DNA duplex (116 in Figure 7) which was
purified by 15% polyacrylamide gel electrophoresis.
This DNA duplex then was enzymatically phosphorylated
using T4 polynucleotide kinase and [y_p32]ATP following
established procedure.
The DNA fragment of 46 bp which runs from the
above-described H~aII site to the PvuII site can either
be constructèd synthetically or obtained from the
original pBP348 plasmid. Accordingly, one hundred
micrograms of plasmid pBP348 were digested in 400 ~1 of
PvuII buffer with 50 units of PvuII for 2 hours at 37C.
After phenol extraction and ethanol precipitation, the
DNA was dissolved in 400 ~1 of PstI buffer (50mM NaCl,
6mM Tris HCl, pH 7.4, 6mM MgC12, 6mM ~-mercaptoethanol)
with 50 units of PstI for 2 hours at 37C. The DNA
fragments were spread on a 6 percent polyacrylamide gel
and the 135 bp fragment containing the desired ~6 bp
sequence was recovered and purified by standard proce-
dures. One-third of the recovered DNA (eguivalent to
33 ~g) was subjected to limited digestion by 1 unit of
H~aII restriction enzyme in 100 ~1 H~aII buffer (20mM
71~3 -
X-6125C-Canada 39
Tris HCl, pH 7.4, 7mM MgC12, 6mM ~-mercaptoethanol) for
40 minutes at 37C. After heating at 65C for 10
minutes, the DNA fragments were run on a 5 percent
acrylamide gel (acrylamide:bis ratio 19:1) along with an
appropriate size marker. The desired 46 bp fragment
yielded by HPaII partial digestion of the 135 bp frag-
ment (from 112 in Figure 7) was purified by standard
procedures.
Two-tenths microgram of the XbaI-PvuII frag-
ment of plasmid vector (115 in Figure 7), 3.2 picomoles
of synthetic 63 bp fragment (116 in Figure 7) and 0.5
picomoles 46 bp fragment (from 112 in Figure 7) were
incubated in 10 ~1 ligation buffer with 2 units of T4
DNA ligase for 16 hours at 4C. The ligation mixture
was used to transform E. coli JA221 and the resultant
transformants, which thus contained the desired plasmid
pNM789, were selected by ampicillin resistance. The
identity of plasmid pNM789 (117 in Figure 7) was con-
firmed by conventionally screening for the presence of
both the 494 bp PvuII the 109 bp XbaI-PvuII fragments.
E. Final Construction of Plasmid PNM789B
Plasmid pNM789 (117 in Figure 8) requires one
amino acid codon change for complete conversion to
bovine growth hormone. This was accomplished by the
removal of the 28 bp PvuII to BamHI fragment of pNM789
and replace~ent with a synthetic double stranded frag-
ment having the following sequence (118 in Figure 8):
5 CTGTGCCTTCTAG3
3,GACACGGAAGATCCTAG5,
~9~8
X-6125C-Canada _40_
Ten micrograms of pNM789 were digested with 1
unit of Pw II in 200 ~1 PvuII buffer for 5 minutes at
37C. After heating 10 minutes at 65C, the mixture was
diluted to 300 ~1 with the addition of BamHI buffer,
digested to completion with 10 units of BamHI for 1 hour
at 37C, treated with 5 units of alkaline phosphatase
and incubated for 1 hour at 65C. The DNA fragments
were separated on a 1 percent agarose gel and a DNA
fragment (119 in Figure 8) about the size of single cut
plasmid pNM78g was conventionally purified. Two-tenths
microgram of this fragment was ligated with 5 picomoles
of synthetic fragment using 2 units of T4 ligase in
20 ~1 ligase buffer. The ligation was carried out
overnight at 4C. Following transformation, several
plasmids were isolated and screened for the appropriate
P w II (494bp) and XbaI-BamHI (628bp) fragments. Plasmids
comprising the aforementioned fragments co~stituted the
desired plasmid pNM789B (120 in Figure 8).
Example 2
Construction of Plasmid pCZ101 and E. coli K12 RV308/-
pCZ101
A. Isolation of Plasmid pIM-I'-A3
The bacterium E. coli K12/pIM-I'-A3 (NRRL
-
B-15733) was cultured in TY broth (1% tryptone, 0.5%
yeast extract, 0.5% sodium chloride, p~ 7.4) with
50 ~g/ml of kanamycin at 25C according to conventional
~?~917~l8 - -
X 6125C-Canada -41-
microbiological procedures. After the culture was
diluted 1:10 into fresh broth and after 3 hours incuba-
tion at 37C, about .5 ml of the culture was transferred
to a 1.5 ml Eppendorf tube and centrifug~d for about 15
seconds. Unless otherwise indicated, all the manipula-
tions were done at ambient temperature. The resultant
supernatant was carefully removed with a fine-tip
aspirator and the cell pellet was resuspended in about
100 ~1 of freshly prepared lysozyme solution (2 mg/ml)
which contained 2 mg/ml lysozyme, SCmM glucose, 10 mM
EDTA (ethylenediaminetetraacetic acid) and 25 mM Tris-HCl,
pH 8. About 200 ,ul of alkaline SDS (sodium dodecyl sulfate)
solution (0.2N NaOH, 1% SDS) were added and the tube was
gently inverted and then kept at 0C until lysis was
complete (~S minutes). Next, about 150 ~1 of 3M sodium
acetate were added and the contents of the tube mixed
gently by inversion for a few seconds.
The tube was maintained at 0C for at least 60
minutes and then centrifuged for 15 minutes to yield an
almost clear supernatant. The supernatant was trans-
ferred to a second centrifuge tube to which 3 volumes of
cold 100% ethanol were added. After the tube was held
on dry ice ethanol for S minutes, the resultant precipi-
tate was collected by centrifugation (5 minutes) and the
supernatant was removed by aspiration. The collected
pellet was dissolved in 100 ~1 of TE (lOmM Tris HCl, pH
8.0, lmM EDTA) and constituted the desired pIM-I'-A3
plasmid DNA.
P~917~8
X-6125C-Canada -4~-
B. XbaI-BamHI Digestion of Plasmid pNM789B and
generation of the ~0.6 kb XbaI-BamHI Fragment
-
About 5 ~g of plasmid pNM789B DNA in 50 ~1 Hi
Salt buffer* were incubated with 10 units each of BamHI
and XbaI restriction enzymes at 37C for about 1 hour.
After the àddition of 5 ~1 of 3M sodium acetate pH 7.0,
the DNA was precipitated with 2 volumes of 100% ethanol.
The desired DNA digest was dissolved in 100 ~1 of TE
buffer and stored at 0C for future use.
Hi Salt buffer was conventionally prepared with the
following composition:
100 mM NaCl
20 mM Tris HCl, pH 8.0
10 mM MgCl2
5 mM ~-mercaptoethanol
C. XbaI-BamHI ~igestion of Plasmid pIM-I'-A3
The desired digestion was carried out in
substantial accordance with the procedure of Example 2B
except that plasmid pIM-I'-A3, rather than plasmid
pNM789B, was used. The desired DNA was dissolved in
about 100 ~1 of TE buffer and stored at 0c. for future
use.
D. Liqation and Transformation
About 1 ~g of the plasmid pIM-I'-A3 digest,
1 ~g of the plasmid pNM789B XbaI-BamHI digest, 40 ~1
water, 5 ~1 (5mM) ATP, 5 ~1 ligation mix* a~d 5 units T4
1?~917~8 - -
X-6125C-Canada 43
DNA ligase were incubated at 20C for about 2 hours.
After incubation at 65C for 2 minutes followed by
cooling on ice, the resultant ligation mixture was used
to transform, in substantial accordance with the trans-
formation procedure of Wensink, 1974, Cell 3:315, E.
coli Kl2 RV308 on TY plates (1% tryptone, 0.5% yeast
extract, 0.5% sodium chloride, 1 5% agar, pH 7.4)
containing 50 ~g/ml of kanamycin. Bacterial strain E.
c _ K12 RV308 has been deposited and made part of the
permanent stock culture collection of the Northern
Regional Research Laboratory, Peoria, Illinois, from
which it is available to the public under the accession
number NRRL B-15624.
Some of the resultant transformants, as
conventionally shown by agarose gel electrophoresis
(Maniatis et al., 1982) and other tests, contained only
the desired ~10.8 kb plasmid. SUch a transformant,
herein designated as E. coli K12 RV308/pCZ101, was
selected, plated on TY agar containing appropriate
antibiotics and then cultured using conventional micro-
biological techniques. The resultant cells were used to
isolate plasmid pCZ101 in substantial accordance with
the procedure of Example 2A.
Ligation mix can be prepared with the following
composition:
500 mM Tris-HC1, pH 7.8
200 mM Dithiothreitol
100 mM MgCl2
~91~18
X-6125C-Canada _44_
Exam~le 3
Construction of Plasmid pCZ1920 and E. coli K12 RV308/-
920
A. Construction of the ~10.2 kb BamHI-XbaI Fragment of
Plasmid Pczlol
The desired fragment was constructed in
substantial accordance with the teaching of Example 2B
except that plasmid pCZ101, rather than plasmid pNM789B,
was used. The desired ~10.2 kb BamHI-XbaI restriction
fragments were conventionally separated and isolated by
agarose gel electrophoresis (Maniatis et al., 1982) and
then dissolved in about 100 ~1 of TE buffer and stored
at 0C for future use,
B. Construction of the ~0.6 kb BamHI-HqiAI Fragment of
Plasmid PCZ101
2~
The desired fragment was constructed in
substantial accordance with the teaching of Example 2B
except that plasmid pCZ101 and HqiAI restriction enzyme,
rather than plasmid pNM789B and XbaI restriction enzyme,
were respectively used. The desired ~0.6 kb BamHI-
HqiAI restriction fragments were conventionally separat-
ed and isolated by agarose gel electrophoresis (Maniatis
et al., 1982) and then dissolved in about 100 ~1 of TE
buffer and stored at 0C for future use.
~9~
X-6125C-Canada -45-
C. Construction of the DNA Linker Sequence
5' CTAGAGC;GTATTMTA ATG AAA GGG AAT TCT ATG GCC TTC CCA GCC
.. I........ .,. .. , .. , ., ... ... I.. ... ... ~.I
5 3 ' TCCCATAATTAT TAC TTT CCC TTA AGA TAC CGG AAG GGT CGG
ATG TCC TTG TCC GGC CTG TTT GCC AAC GCT GTGCT 3 '
.,. .., .-- ,.. ,,, .., ... ~.. ... .I. I
TAC AGC; AAC AGG CCG GAC AAA CGG TTG CGA C 5'
The desired linker sequence was conventionally
synthesized by the modified phosphotriester method in
substantial accordance with the teaching of Itakura et
al., 1977 and Crea et al., 1978. The aforementioned
synthesis method is also specifically illustrated in
Example 1.
D. Liaation and Transformation
About 20 picomoles of the DNA linker of
Example 3C, 1 ~g of the plasmid pCZ101 ~10.2 kb BamHI-
XbaI fragment and 0.5 ~g of the plasmid pCZ101 ~0.6 kb
BamHI-HqiAI fragment were ligated and the resultant
plasmid used to transform E. coli K12 RV308 in substan-
tial accordance with the teaching of Example 2D.
Some of the resultant transformants, as
conventionally shown by agarose gel electrophoresis
(Maniatis et al., 1982) and other tests, contained only
the desired ~10.8 kb plasmid. Such a transformant,
herein designated as E. coli K12 RV308/pCZ1920, was
selected, plated on TY agar containing appropriate
antibiotics~and then cultured using conventional micro-
biological techniques. The resultant cells were shown,
by SDS gel electrophoresis, RIA and other tests, to
9~718
X-6125C-Canada -46-
express the aforedefined MET-LYS-GLY-ASN-SER-MET-ALA-bGH
derivative at high levels. Because plasmid pCZ1920
contains a thermoinducible runaway replicon, maximum
expression of the desired bGH derivative product occurs
S at culture temperatures of about 37C.
Example 4
Construction of Plasmid pJRl and E. coli K12 RV308/-
1 0 PJRl
The desired constructions are made in substan-
tial accordance with the teaching of Example 3 except
that the DNA linker sequence
S' CTAGAGGGTATTAATA ATG TTT CCA GCC ATG GCT CTA TCT GGT
,........... ... ... ... ... ... ... ... -. .-
3' TCCCATAATTAT TAC AAA GGT CGG TAC CGA GAT AGA CCA
CTG TTT GCC AAC GCT GTGCT 3'
... ... ... ... ... .
GAC AAA CGG TTG CGA C 5'
is substituted for the linker sequence of Example 3C.
The above-specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pJR1, are plated on TY agar con-
taining appropriate antibiotics and then conventionally
cultured for subsequent production and isolation of
~X917~8
X-6125C-Canada _47_
plasmid pJRl. The transformants are also shown, by SDS
gel electrophoresis, RIA and other tests, to express the
aforedefined MET-PHE-PRO-ALA-MET-ALA-R2 bovine growth
hormone derivative at high levels. Beca~se plasmid pJRl
contains a thermoinducible runaway replicon, maximum
expression of the desired bGH derivative product occurs
at culture temperatures of about 37C.
ExamDle 5
Construction of Plasmid PHI7~4~1
A partial scheme for the construction of
plasmid pHI7~4Al is presented in Figure 15 of the accom-
panying drawings.
A. Construction of Plasmid ~BRHtrP
Plasmid pGMl carries the E. coli tryptophan
operon containing the deletion ~LE1413 (Miozzari, etal., 1978, J. Bacteriology, 1457-1466) and hence ex-
presses a fusion protein comprising the first 6 amino
acids of the trp leader and approximately the last third
of the tr~ E polypeptide (hereinafter referred to in
conjunction as LE'), as well as the trP D polypeptide in
its entirety, all under the control of the trP promoter-
operator system. E. coli K12 W3110tna2trp-~102/pGMl has
been deposited with the American Type Culture Collection
(ATCC No. 31622) and pGMl may be conventionally removed
from the strain for use in the procedures described
below.
~917~8
X-6125C-Canada -48-
A~out 20 ~g of the plasmid were digested with
the restriction enzyme PvuII which cleaves the plasmid
at five sites. The gene fragments were next combined
with EcoRI linkers (consisting of a self complementary
S oligonucleotide of the sequence: pCATGAATTCATG) pro-
viding an EcoRI cleavage site for later cloning into a
plasmid containing an EcoRI site. The 20 ~g of DNA
fragments obtained from pGMl were treated with lO units
T4 DNA ligase in the presence of 200 picomoles of the
5'-phosphorylated synthetic oligonucleotide pCATGAATTCATG
and in 20 ~1 T4 DNA ligase buffer (20 mM Tris, pH ~.6,
O.5 mM ATP, lO mM MgC12, 5 mM dithiothreitol) at 4C
overnight. The solution was then heated 10 minutes at
70C to halt ligation. The linkers were cleaved by
EcoRI digestion and the fragments, now with EcoRI ends,
were separated using 5 percent polyacrylamide gel
electrophoresis (hereinafter "PAGE"). The three largest
fragments were isolated from the gel by first staining
with ethidium bromide and then locating the fragments
with ultraviolet light and cutting from the gel the
portions of interest. Each gel fragment, with 300 ~l
O.lxTBE, was placed in a dialysis bag and subjected to
electrophoresis at lO0 v for one hour in O.lxTBE buffer
(TBE buffer contains: 10.8 gm Tris base, 5.5 gm boric
acid, .09 gm Na2EDTA in l liter H2O). The aqueous
solution was collected from the dialysis bag, phenol
extracted, chloroform extracted, and made 0.2M with
respect to sodium chloride. The DNA was then recovered
in water after ethanol precipitation. The tr~ pro-
moter/operator-containing fragments with EcoRI sticky
~9~7~8
X-6125C-Canada ~49~
ends were identified by insertion into a tetracycline
sensitive plasmid which, upon promoter/operator inser-
tion, becomes tetracycline resistant. All DNA fragment
isolations hereinafter described in this example are
performed using PAGE followed by the electroelution
method described above.
B. Construction of Plasmid pBRHtrp ExPressinq Tetra-
cycline Resistance Under the Control of the trp
PromoterjOperator and Identification and AmPlifi-
cation of the tr~ Promoter/Operator-Containing DNA
Fraqment Isolated in 'A' above.
Plasmid pBRHl, (Rodriguez, et al., 1979,
Nucleic Acids Research 6, 3267-3287 and ATCC No. 37070)
expresses ampicillin resistance and contains the gene
for tetracycline resistance but, there being no associ-
ated promoter, does not express that resistance. Cells
harboring the plasmid are accordingly tetracycline
sensitive. By introducing a promoter/operator system in
the EcoRI site, the plasmid will express tetracycline
resistance.
Plasmid pBRHl was digested with EcoRI. The
enzyme was removed by phenol extraction followed by
chloroform extraction and then the DNA was resuspended
in water after ethanol precipitation. The resulting DNA
was, in separate reaction mixtures, combined with each
of the three DNA fragments obtained in Example 5A above
and ligated with T4 DNA ligase as previously described.
~9~8
~-6125C-Canada -so-
The DNA present in the reaction mixture was used to
transform competent E. coli K12 294 (NRRL B-15625) by
standard techniques (Hershfield Q al., 1974, Proc. Nat.
Acad: Sci. USA 71:3455-3459) and the bacteria were then
plated on L~ plates (Maniatis et al., 1982) containing
20 ~g/ml ampicillin and 5 ~g/ml tetracycline.
Several tetracycline-resistant colonies were
selected and the plasmid DNA was isolated and designated
pBRHtrp. The presence of the desired fragment was
confirmed by restriction enzyme analysis. Plasmid
p8RHtrp expresses ~-lactamase, imparting ampicillin
resistance, and contains a DNA fragment which includes
the trP promoter/operator. The DNA fragment also codes
for a first protein, (designated LE'), comprising a
fusion of the first six amino acids of the trP leader
and approximately the last third of the rp E poly-
peptide, a second protein (designated D'), corresponding
to approximately the first half of the ~ D poly-
peptide, and a third protein, coded for by the tetra-
cycline resistance gene.
C. Construction of Plasmid pSOM7~2
Plasmid pBRHtrp was digested with EcoRI
restriction enzyme and the resulting fragment, isolatedby PAGE and electroelution, was combined with EcoRI-
digested plasmid pSOMll (Itakura et al., 1977, Sci.
198:1056, U.S. Patent No. 4,366,246, G.B. Patent Publi-
cation No. 2,007,676A). The mixture was ligated with T4
DNA ligase and the resulting DNA transformed into E.
~91~8
X-6125C-Canada -51-
coli K12 294 as previously described. Transformant
bacteria were selected on ampicillin-containing plates
and the resulting ampicillin-resistant colonies were
screened by colony hybridization (Gruenstein et al.,
1975, Proc. Nat. Acad. Sci. USA 72:3951-3965). The
promoter/operator-containing fragment, isolated from
pBRHtrp and then radioactively labelled with 32p, was
used as a probe in the above procedure. Several colo-
nies were shown to be positive by colony hybridization
and were therefore selected. Plasmid DNA was isolated
and the orientation of the inserted fragments was
determined by restriction ana}ysis using enzymes 8glII
and BamHI in double digestion. Colonies containing the
desired plasmid with the trP promoter/operator fragment
in the proper orientation were grown in LB medium
containing 10 ~g/ml ampicillin. The desired plasmid was
designated pSOM7~2 and was used for subseguent construc-
tions described below.
D. Construction of Plasmid ptrp24
1. Construction of a Gene Fraoment Codinq for the
Distal Re~ions of the LE' PolY~eDtide With
~II and EcoRI Restriction Sites Respectively
at the 5' and 3' Ends of the Codinq Strand
Plasmid pSOM7~2 was HindIII digested followed
by digestio~ with lambda exonuclease (a 5' to 3' exo-
nuclease) under conditions chosen so as to digest beyond
the BalII restriction site within the LE' encoding
~9171~3
X-6125C-Canada -52-
region. About 20 ~g of HindIII-digested pSOM7~2 was
dissolved in buffer (20mM glycine buffer, pH 9.6, lmM
MgC12, lmM ~-mercaptoethanol). The resulting mixture
was treated wi~h S units of lambda exonuclease for 60
minutes at room temperature. The reaction mixture
obtained was then phenol extracted, chloroform extract-
ed, and ethanol precipitated.
To create an EcoRI residue at the distal end
of the LE' gene fragment, a primer 32pCCTGTGCATGAT was
synthesized by the improved phosphotriester method (Crea
et al., 1978) and hybridized to the single stranded end
of the LE' gene fragment resulting from lambda exonuclease
digestion. The hybridization was performed by dissolv-
ing 20 ~g of the lambda exonuclease-treated HindIII
digestion product of plasmid pSOM7~2 in 20 ~1 H2O and
combining with 6 ~1 of a solution containing approxi-
mately 80 picomoles of the 5'-phosphorylated oligonu-
cleotide described above. The synthetic fragment was
hybridized to the 3' end of the LE' coding sequence and
the remaining single strand portion of the LE' fragment
was filled in by Klenow Polymerase I using dATP, dTTP,
dGTP and dCTP. Klenow Polymerase I is the fragment
obtained by proteolytic cleavage of DNA Polymerase I.
It contains the 5' ~ 3' polymerizing activity, the
25 3 ' ~ 5 ' exonucleolytic activity, but not the 5' ~ 3'
exonucleolytic activity of the parental enzyme
(Kornberg, 1974, W. H. Freeman and Co., SFO, 98).
The reaction mixture was thus heated to 50C
and let cool slowly to 10C, whereafter 4 ~1 of Klenow
enzyme were added. After 15 minutes incubation at room
.~9~7~
X-6125C-Canada _53_
temperature, followed by 30 minutes incubation at 37C,
the reaction was stopped by the addition of S ~1 of 0.25
molar EDTA. The reaction mixture was phenol extracted,
chloroform extracted, and ethanol precipi~ated. The DNA
5 was subsequently cleaved with the restriction enzyme
BqlII and the fragments were separated by PAGE. An
autoradiogram obtained from the gel revealed a 32p
labelled fragment of the expected length of approximate-
ly 470 bp, which was recovered by electroelution. As
outlined, this fragment LE'(d) has a BqlII terminus and
a blunt end coincidin~ with the beginning of the primer.
2. Construction of Plasmid pTh~l
Plasmid pTh~l was constructed by inserting a
synthesized gene for thymosin alpha 1 into plasmid
pBR322. The synthesis of the thymosin alpha 1 coding
DNA involves the synthesis and subsequent ligation of
the 16 oligonucleotides (Tl through T16) that are
indicated by the double headed arrows in Figure 11 of
the accompanying drawings. A met ATG was inserted at
the N-terminus and the 5' ends were designed with
single-stranded cohesive termini to facilitate joining
to plasmids cleaved with EcoRl and BamHl. As can be
readily appreciated, the BqlII site in the center of the
gene assists in the analysis of recombinant plasmids.
Oligodeoxyribonucleotides Tl to T16 were
synthesized by the modified phosphotriester method of
Itakura et al., 1977 and Crea et al., 1978. The various
oligodeoxyribonucleotides are shown below in Table 1.
~917~ ~ ~
X-6125C-Canada -54-
Table 1
SYNTHETIC OLIGONUCLEOTIDES FOR THYMOSINal GENE
HPLC
Analysis
Retention
Time
ComPound Sequence Lenqth (min)*
Tl A-A-T-T-C-A-T-G-T-C 10 17.4
T2 T-G-A-T-G-C-T-G-C-T-G-T-T-G-A 15 24.3
T3 T-A-C-T-T-C-T-T-C-T-G-A 12 20.3
T4 G-A-T-T-A-C-T-A-C-T-A-A-A 13 22.0
lS T5 G-C-A-G-C-A-T-C-A-G-A-C-A-T-G 15 24.8
T6 G-A-A-G-T-A-T-C-A-A-C-A 12 20.1
T7 A-G-T-A-A-T-C-T-C-A-G-A-A 13 22.6
T8 A-A-G-A-T-C-T-T-T-A-G-T 12 20.2
Tg G-A-T-C-T-T-A-A-G-G-A-G 12 20.4
Tlo A-A-G-A-A-G-G-A-A-G-T-T 12 21.1
Tll G-T-C-G-A-A-G-A-G-G-C-T 12 20.5
T12 G-A-G-A-A-C-T-A-A-T-A-G 12 20.4
T13 C-T-T-C-T-T-C-T-C-C-T-T 12 19.9
T14 T-T-C-G-A-C-A-A-C-T-T-C 12 20.5
T15 G-T-T-C-T-C-A-G-C-C-T-C 12 20.2
T16 G-A-T-C-C-T-A-T-T-A 10 17.2
*
at ambient temperature
X-6125C-Canada ~9~718
The above synthesis is typified by the follow-
ing procedure for fragment T15 as summarized in Figure 12
of the accompanying drawings. Various nucleotide
fragments that are used in the synthesis of T15 are
numerically designated in the Figure. The abbreviations
employed are as follows: TPSTe, 2,4,6-triisopropyl-
benzenesulfonyltetrazole; BSA, benzene sulfonic acidi
TLC, thin layer chromatography; HPLC, high performance
liquid chromatography; DMT, 4,4'-dimethoxytrityl; CE,
2-cyanoethyl; R, p-chlorophenyl; Bz, benzoyl; An,
anisoyl; iBu, isobutyryl; Py, pyridine; AcOH, acetic
acid; ~t3N, triethylamine.
The fully protected trideoxyribonucleotides 4
(85 mg, 0.05 mmol) and 2 (180 mg, 0.1 mmol) were deblocked
at the 5' hydroxyls by treatment with 2% BSA in 7:3
(v/v) chloroform/methanol (10 and 20 ml, respectively)
for 10 minutes at 0C. Reactions were stopped by
addition of saturated aqueous ammonium bicarbonate
(2 ml), extracted with chloroform (25 ml) and washed
with water (2 x 10 ml). The organic layers were dried
(magnesium sulfate), concentrated to small volumes
(about 5 ml) and precipitated by addition of petroleum
ether (35-60C fraction). The colorless precipitates
were collected by centrifugation and dried in a desiccator
ln vacuo to give 6 and 8, respectively, each homogeneous
by silica gel tlc (Merck 60 F254, chloroform/methanol,
9:1)-
Trimers 1 and 3 (270 mg, 0.15 mmol; 145 mg,
0.075 mmol) were converted into their phosphodiesters (5
and 7) by treatment with triethylamine/pyridine/water
P~9~
X-6125C-Canada -56-
(1:3:1, v/v, 10 ml) for 25 minutes at ambient tempera-
ture. Reagents were removed by rotary evaporation and
the residues dried by repeated evaporations with anhy-
drous pyridine ~3x 10 ml). Trimer 8 (0.05 mmol) and
trimer 7 were combined with TPSTe (50 mg, 0.15 mmol) in
anhydrous pyridine (3 ml) and the reac~ion mixture left
ln vacuo at ambient temperature for two hours. TLC
analysis showed that 95% of the trimer 8 had been
converted into hexamer product (visualized by detection
of the DMT group by spraying with 10% aqueous sulfuric
acid and heating at 60C). The reaction was guenched by
addition of water (1 ml) and the solvent evaporated
under reduced pressure. After removal of pyridine by
coevaporations with toluene, the hexamer was deblocked
at the 5' position with 2% BSA (8 ml) as described above
for trimers 4 and 2. The product (10) was purified on a
silica gel column (Merck 60 H, 3.5 x 5 cm) by step
gradient elution with chloroform/methanol (98:2 to ~5:5,
v/v). Fractions containing product 10 were evaporated
to dryness. Similarly, trimer 5 was coupled to 6 and
the fully protected product directly purified on silica
gel. This latter compound was deblocked at the 3' end
by triethylamine/pyridine/water as described above to
give fragment 9.
Finally, hexamers 9 and 10 were coupled in
anhydrous pyridine (2 ml~ with TPSTe (75 mg, 0.225 mmol)
as the condensing agent. Upon completion (4 hours,
ambient temperature) the mixture was rotary evaporated
and the residue chromatographed on silica gel. Product
11 (160 mg) was obtained by precipitation with petroleum
~91~ -
X-6125C-Canada -57-
ether and appeared homogeneous on TLC. A portion of
compound 11 (20 mg) in pyridine (0.5 ml) was completely
deblocked by treatment with concentrated ammonium
hydroxide (7 ml, 8 hours, 50C) and subsequent treatment
in 80% acetic acid (15 minutes, ambient temperature).
After evaporation of acetic acid, the solid residue was
dissolved in 4% aqueous ammonium hydroxide (v/v, 4 ml)
and extracted with ethyl ether (3x2 ml). The agueous
phase was concentrated to 1-2 ml and a portion applied
to HPLC for purification of 12. The fractions corre
sponding to the major peak were pooled (ca. 2 A254
units) and concentrated to about 5 ml. The final
product 12 was desalted on ~io-gel P-2 (1.5 x 100 cm) by
elution with 20% aqueous ethanol, reduced to dryness and
resuspended in water (200 ~1) to give a solution of
A254 = 10. The sequence of 12 was confirmed by two-
dimensional seguence analysis.
The complete thymosin alpha 1 gene was assem-
bled from the 16 synthetic oligo-nucleotides by methods
previously described in detail for somatostatin (Itakura
et al., 1977), insulin (Goeddel et al., 1979), and
growth hormone (Goeddel et al., 1979, Nature 281:544).
Ten microgram quantities of oligonucleotides T2 through
T15 were quantitatively phosphorylated with [y_p32]-ATP
(New England Nuclear) in the presence of T4 polynucleotide
kinase (Goeddel et al, 1979), to give specific activi-
ties of approximately 1 Ci/mmol. Radiolabelled frag-
ments were purified by 20% polyacrylamide/7 M urea gel
electrophoresis and sequences of the eluted fragments
were verified by two-dimensional
*Trademark
~- 612 5C-Canada - 598~7~8
electrophoresis/homochromatography (Jay et al., 1974,
Nucleic Acids Res. 1:331) of partial snake venom di-
gests. Fragments Tl and T16 were left unphosphorylated
to minimize undesired polymerization during subsequent
ligation reactions. These oligonucleotides (2 ~g each)
were assembled in four groups of four fragments (see
figure 13 of the accompanying drawings), by T4 ~NA
ligase using published procedures (Goeddel et al.,
1979). The reaction products were purified by gel
electrophoresis on a 15% polyacrylamide gel containing
7 M urea (Maxam and Gilbert, 1977, Proc. Nat. Acad. Sci.
USA 71:3455). The four isolated products were ligated
together and the reaction mixture resolved by 10%
polyacrylamide gel electrophoresis. DNA in the size
range of the thymosin alpha 1 gene (90-105 base pairs)
was electroeluted.
Plasmid pBR322 (0.5 ~g) was treated with BamHI
and EcoRI restriction endonucleases and the fragments
separated by polyacrylamide gel electrophoresis. The
large fragment was recovered from the gel by electro-
elution and subsequently ligated to the assembled
synthetic DNA (Goeddel et al., Nature 281:544 1979).
This mixture was used to transform E. coli K12 254.
Five percent of the transformation mixture was plated on
LB plates containing 20 ~g/ml ampicillin. The four
~npicillin resistant colonies obtained were sensitive to
tetracycline, suggesting insertion into the tetracycline
resistance gene. Analysis of the plasmids from these
four colonies showed that in each case the plasmid,
designated pTh~l, contained (a) a B~lII site not found
.~91718
~-6125C-Canada _59_
in pBR322 itself, thus indicating the presence of the
thymosin alpha 1 gene as shown in Figure 11, and (b) a
fragment of approximately 105 base pairs generated by
BamHI/EcoRI cleavage. The construction route for
plasmid pThal (not drawn to scale), is presented in
Figure 13 of the accompanying drawings wherein the heavy
dots indicate 5'-phosphate groups.
3. Reaction of Treated PTh~l and LE'(d) Fraqment
The plasmid pThal contains a gene specifying
ampicillin resistance and a structural gene specifying
thymosin alpha 1 cloned at its 5' coding strand end into
an EcoRI site and at its 3' end into a BamHI site. The
thymosin gene contains a BalII site as well. To create
a plasmid capable of accepting the LE'(d) fragment
prepared above, pTHal was EcoRI digested followed by
Klenow polymerase I reaction with dTTP and dATP to blunt
the E RI residues. BqlII digestion of the resulting
product created a linear DNA fragment containing the
gene for ampicillin resistance and, at its opposite
ends, a sticky BqlII residue and a blunt end. The
resulting product could be recircularized by reaction
with the LE'(d) fragment containing a BqlII sticky end
and a blunt end in the presence of T4 ligase to form the
plasmid ptrp24. In doing so, an EcoRI site is recreated
at the position where blunt end ligation occurred.
~9
-6125C-Canada -60-
E. Construction of Plasmid pSOM7~2~4
Successive digestion of ptrp24 with BqlII and
EcoR~, followed by PAGE and electroelution, yields a
fragment having codons for the LE'(d) polypeptide with a
BalII sticky end and an EcoRI sticky end adjacent to its
3' coding terminus. The LE'(d) fragment can be cloned
into the BqlII site of plasmid pSOM7~2 to form an LE'
polypeptide/somatostatin fusion protein expressed under
the control of the tryptophan promoter/operator. To do
so requires (1) partial EcoRI digestion of pSOM7~2 in
order to cleave the EcoRI site distal to the tryptophan
promoter/operator, and (2) proper choice of the primer
sequence in order to properly maintain the codon reading
frame, and to recreate an EcoRI cleavage site.
Thus, 16 ~g of plasmid pSOM7~2 were diluted
into 200 ~1 of buffer containing 20 mM Tris, pH 7.5,
5 mM MgC12, O.02% NP40 detergent, and 100 mM NaCl, and
treated with 0.5 units EcoRI. After 15 minutes at 37C,
the reaction mixture was phenol extracted, chloroform
extracted, ethanol precipitated, and subsequently
digested with BqlII. The larger resulting fragment was
isolated by the PAGE procedure followed by electroelution.
This fragment contains the codons "LE'(p)" for the
proximal end of the LE' polypeptide, i.e., those up-
stream from the BqlII site. This fragment was next
ligated to the above LE'(d) fragment in the presence of
T4 DNA ligase to form the plasmid pSOM7~2~4, which upon
transformation into E. coli K12 294, efficiently pro-
duced a fusion protein consisting of the fully
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A-6125C-Canada -61-
reconstituted LE polypeptide and somatostatin under the
control of the tryptophan promoter/operator.
F. Construction of Linear DNA Having a PstI Residue
at the 3' end and a BqlII Residue at its 5' End
Boundinq a Gene SPecifyinq TetracYcline Resistance
Plasmid pBR322 was HindIII digested and theprotruding HindIII ends were digested with S1 nuclease.
The Sl nuclease digestion involved treatment of 10 ~g of
~indIII_cleaved pBR322 in 30 ~1 Sl buffer (0.3M NaCl,
1 mM ZnC12, 25 mM sodium acetate, pH 4.5) with 300 units
Sl nuclease for 30 minutes at 15C. The reaction was
stopped by the addition of 1 ~1 of 30X Sl nuclease stop
solution (0.8M tris base, 50 mM EDTA). The mixture was
phenol extracted, chloroform extracted, ethanol precipi-
tated, and then EcoRI digested as previously described.
The resulting ragment, obtained by the PAGE procedure
followed by electroelution, has an EcoRI sticky end and
a blunt end whose coding strand begins with the nucleotide
thymidine. The Sl-digested HlndIII residue beginning
with thymidine can be joined to a Klenow Polymerase
I-treated BqlII residue so as to reconstitute the BqlII
restxiction site upon ligation.
Therefore plasmid pSOM7~2, prepared in
Example 5C, was BqlII digested and the resulting 8qlII
sticky ends were made double stranded by treatment with
Klenow polymerase I using all four deoxynucleotide
triphosphates. EcoRI cleavage of the resulting product,
followed by PAGE and electroelution of the small
~?,917~8
~-6125C-Canada -62-
fragment, yielded a linear piece of DNA containing thetryptophan promoter/operator and codons of the LE'
"proximal" seguence upstream from the BqlII site
("LE'(p)l'). The product had an EcoRI end and a blunt
end resulting from filling in the BqlII slte. However,
the BglII site is reconstituted by ligatlon of the blunt
end to the blunt end of the above Sl-digested HindIII
fragment. Thus, the two fragments were ligated in the
presence of T4 DNA ligase to form the recircularized
plasmid pHKY10 which was propagated by transformation
in~o competent E. coli K12 294 cells. Tetracycline
resistant cells bearing the recombinant plasmid p~KY10
were selected and the plasmid DNA extracted. Digestion
with BqlII and PstI, followed by isolation by the PAGE
procedure and electroelution of the large fragment,
yielded the desired linear piece of DNA having PstI and
~II sticky ends. This DNA fragment, thus produced
from pHKY10, contains the origin of replication and,
therefore, is useful as a component in the construction
of plasmid pHI7~4~1 in which both the genes coding for
the trP LE' polypeptide fusion protein and the tetra-
cycline resistance are controlled by the tr~ promoter/
operator.
G. Constructîon of Linear DNA Having the
Promoter/OPerator
Plasmid pSOM7Q2~4, prepared in Example 5E, was
subjected to partial EcoRI digestion followed by PstI
digestion. The resulting fragment contained the trp
~L~ `?1917~3
X-6125C-Canada -63-
promoter/operator and was isolated by the PAGE procedure
followed by electroelution. Partial EcoRI digestion was
necessary to obtain a fragment which was cleaved adja-
cent to the 5' end of the somatostatin gene but not
cleaved at the EcoRI site present between the ampicillin
resistance gene and the trP promoter/operator. Ampicillin
resistance lost by the PstI cut in the ampicillin
resistance gene can be restored upon ligation with the
final pHKY10 linear DNA derivative produced in Example 5F
above.
H. PreParation of SYnthetic Gene Codinq for the
32 N-Terminal Amino Acids of Proinsulin
A series of 18 oligonucleotides, shown in
Table 2, were prepared as a first step in constructing a
gene coding for the first 32 amino acids of proinsulin.
The nucleotide sequence of the entire gene ultimately
constructed is shown in Figure 14.
1~9~8
X-6125C-Canada -64-
Table 2
SYnthetic Oliqonucleotides For Human Proinsulin
ComDound Sequence
Hl AATTCATGTT
H2 CGTCAATCAGCA
H3 CCTTTGTGGTTC
H4 TCACCTCGTTGA
H5 TTGACGAACATG
H6 CAAAGGTGCTGA
H7 AGGTGAGAACCA
H8 AGCTTCAACG
Bl AGCTTTGTAC
B2 CTTGTTTGCGGT
B3 GAACGTGGTTTC
B4 TTCTACACTCCT
B5' MGACTCGCC
B6 AACAAGGTACAA
B7 ACGTTCACCGCA
B8 GTAGAAGAAACC
B9 AGTCTTAGGAGT
B10' GATCCGGCG
~ ?~91~
X-6125C-Canada -65-
The synthetic nucleotides are shown between
brackets and are underlined in Figure 14. These
olignucleotides were synthesized by the conventional
method of Crea et al., 1978, J. Biol. Chem. 250:4592
and Itakura et al., 1975, J. Am. Chem. Soc. 97:7327.
Some of the oligonucleotides were used for constructing
a gene for the human insulin B chain previously described
by Crea, et al.,,l978 and Goeddel, et al, 1979. Two
oligonucleotides (B5' and B10') incorporate HpaII and
terminal BamHI and EcoRI restriction sites. The terminal
sites are particularly useful for purposes of cloning.
The eight oligonucleotides Hl-H8, used previ-
ously for constructing the left half of the human
insulin B chain gene (Goeddel, et al., 1979), contain
the coding sequence for the 1-13 amino acids of the B
chain ~ene and an additional N-terminal methionine. The
right half of the B chain gene was constructed from
oligonucleotides Bl, B2~ B3~ B4~ Bs ~ 6 7 8 9
Blo by ligation using T4 DNA ligase in substantial
accordance with the teaching of Goeddel, et al., 1979.
The resultant gene fragment codes for the 14-30 amino
acid units of the human insulin B chain and the first
arginine of the bridging chain. A HPaII restriction
site is incorporated into the gene sequence in the same
reading frame and location as the HDaII site in the
human insulin gene. After purification of the ligated
gene fragment by polyacrylamide gel electrophoresis and
after elution of the largest DNA band, the fragment was
inserted into HindIII-BamHI-cleaved plasmid pBR322. The
resultant plasmid, designated pB3, was inserted into E.
~?.~
X-6125C-Canada -66-
coli K12 294 by transformation. The plasmid conferred
resistance to antibiotics ampicillin and tetracycline
and was found to contain the desired nucleotide
sequence.
Two fragments, a 58 base pair HindIII-BamHI
fragment of pB3 and a 46 base pair EcoRI-HindIII frag-
ment of pBHl (disclosed in Goeddel, et al., 1979), were
ligated to produce a fragment having EcoRI and BamHI
termini. This fragment was conventionally ligated into
EcoRI and BamHI restricted plasmid pBR322 and the
resultant plasmid, designated pIB3, was then cloned into
E. coli K12 294. After conventional amplification and
isolation, plasmid pIB3 was digested with EcoRI and
HPaII to produce a synthetic gene fragment (Fragment 1,
Figure 15) that codes for the N-terminal proinsulin
amino acids preceded by a methionine. The synthetic
gene was isolated conventionally by polyacrylamide gel
electrophoresis.
I. Isolation of cDNA Codinq for the 50 C-Terminal
Amino Acids of Human Proinsulin
The scheme for obtaining the desired cDNA is
presented in Figure 16 of the accompanying drawings. In
accordance therewith, the DNA sequence pCCGGATCCGGTTT18T,
which contained both a BamHI recognition sequence and a
3' polythymidylic acid tract of approximately 20 resi-
dues, was synthesized and used to prime AMV reverse
transcriptase for cDNA synthesis. The primer was
prepared using terminal deoxynucleotidyl transferase
~9~18
X-612SC-Canada -67~
(Enzo Biochem, 200 units) with 1 ~mol of the BamHI
decanucleotide in a reaction volume of 0.6 ml containing
1.5 x 10 4 ~mol TTP. The reaction was conducted at 37C
for 1 hour in the buffer system of Chang, et al., 1978,
Nature 275:617.
Human insulinoma tissue was provided by the
Institute fur Diabetesforschung, Muenchen, west Germany.
Those skilled in the art understand that human insulinoma
tissu~ is readily available and can be obtained from a
number of other sources in the medical community. Poly
A mRNA (2.5 ~g) of the human insulinoma tissue was
isolated in substantial accordance with the procedure of
Ullrich, _ al., 1977, Science 196:1313 and then con-
verted to double stranded cDNA in substantial accordance
with the teaching of Wickens, et al., 1978, J. Biol.
Chem. 253:2483. Thus, a reaction volume of 80 ~1 con-
taining lS mM Tris HCl ~pH 8.3 at 42C), 21 mM KCl,
8 mM MgC12, 30 mM B-mercaptoethanol, 2 mM of the primer
dCCGGATCCGGTT18T, and 1 mM dNTPs was preincubated at
0C. After addition of AMV reverse transcriptase, the
mixture was incubated for 15 minutes at 42C. The
resultant RNA/DNA was then denatured using conventional
procedures.
The complementary cDNA strand was synthesized
in a reaction volume of 150 ~1 containing 25 mM Tris HCl,
pH 8.3, 35 mM KCl, 4 mM MgC12, lS mM ~-mercaptoethanol
1 mM dNTPs and 9 units of Klenow Polymerase I. The
mixture was incubated at 15C for 90 minutes followed by
15 hours at 4C. Sl nuclease digestion was then
performed for 2 hours at 37C using 1000 units of S1
~91~
X-6125C-Canada -68-
nuclease (Miles Laboratories, Elkhart, Indiana) in
substantial accordance with the teaching of Wickens, et
al., 1978. The double stranded cDNA (0.37 ~g) was
electrophoresed on an 8% polyacrylamide ~el and DNA
fragments larger than 500 base pairs were eluted.
Oligodeoxycytidylic acid residues were added to the 3'
ends of the fragments using terminal deoxynucleotidyl
transferase in substantial accordance with the procedure
of Maizel, 1971, Meth. Virol. 5:180. The dC tailed cDNA
fragments were then annealed to pBR322 that had first
been digested with PstI restriction enzyme and then
tailed with deoxyguanidylic acid using terminal deoxy-
nucleotidyl transferase. The resulting plasmids were
used to transform E. coli K12 294 and the resultant
cells plated on LB and TET medium. Colonies resistant
to tetracycline but sensitive to ampicillin were isolat-
ed and screened for plasmids that had three PstI re-
striction sites. Such a restriction pattern is
indicative of the gene for proinsulin, Sures, et al.,
1980, Science 208:57. One plasmid, designated as
pHI104, contained a 600 base pair insert, gave the
anticipated PstI restriction pattern and contained a
BamHI site between the 3' polyA and the polyGC intro-
duced during the cDNA preparation. Some of the nucleo-
tide sequence of the insert is shown in Figure 14.
~9~
X-6125C-Canada -69-
J. AssemblY of a Gene Coding for Human Proinsulin
The scheme used for assembling a gene coding
for human proinsulin is shown in Figure 15 of the
accompanying drawings.
The synthetic gene segment coding for the
first 31 amino acids of proinsulin, fragment 1 in
Figure 15, was recovered from 50 ~g of plasmid pIB3
using the restriction endonucleases EcoRI and H~aII as
described above. This fragment also contains the ATG
sequence for methionine in place of the "presequence" of
preproinsulin.
The cDNA gene segment coding for amino acids
32-86, as well as the translation stop signal and the 3'
untranslated region of the mRNA, was recovered from
40 ~g of plasmid pHI104 by treatment first with BamHI
and then H~alI restriction enzymes. The two fragments
were isolated by polyacrylamide gel electrophoresis
followed by electroelution. The gene fragments were
joined by treatment with T4 DNA ligase in 20 ~1 ligase
buffer at 4C for 24 hours. The mixture was diluted
with 50 ~1 H20, conventionally extracted with each of
phenol and chloroform and then precipitated with
ethanol.
The resulting DNA was treated with BamHI and
EcoRI restriction enzymes to regenerate these sites and
remove gene polymers. The assembled proinsulin gene was
isolated by polyacrylamide gel electrophoresis and
ligated (using T4 DNA ligase) to EcoRI and BamHI digest-
ed plasmid pBR322. The resulting DNA was used to
~91~8
X-6125C-Canada -70-
transform E. coli K12 294 and then the resultant colo-
nies were screened for tetracycline sensitivity and
ampicillin resistance. Plasmid pHI3, isolated from one
such colony, contained the desired proinsulin gene which
S was subsequently characterized by nucleotide sequence
analysis.
K Construction of a Plasmid for ExPression of a
.
Chimeric Protein Containinq Human Proinsulin
The complete human proinsulin gene, including
the N-terminal codon that codes for methionine, was
recovered from plasmid pHI3 by treatment with EcoRI and
BamHI restriction enzymes. The desired fragment was
purified by gel electrophoresis and then ligated (using
T4 DNA ligase) ~o the plasmid pSOM7~2~4 PstI-EcoRI
partial digest (prepared in Example SG) and the larger
of the PstI-BqllI fragments of plasmid pHKY10 (prepared
in Example SF). Thus, about 1 ~g of the complete human
proinsulin gene with EcoRI and BamHI termini, 4 ~g of
PstI-EcoRI ~partial) pSOM7~2a4 fragment, and about 1 ~g
of the PstI-BqlII fragment of pHKY10 were ligated at
4C. for 24 hours using T4 DNA ligase in ligation
buffer. The ligated DNA mixture was used to transform
E. coli K12 294. Colonies that grew on both ampicillin
and tetracycline were selected and were found to contain
the desired plasmid pHI7~4~1 and to express a protein of
the molecular weight expected o the trp LE'- proinsulin
fusion. Plasmid pHI7~4~1, which expressed the aforemen-
tioned protein, was completely characterized as to the
~9~
X-6125C-Canada -71-
DNA se~uence and restriction sites of both the incorpo-
rated gene and also the vector. Plasmid pHI7~4Al is
depicted in Figure 15 of the accompanying drawings and
can be selected readily ~ecause of the restoration of
S ampicillin and tetracycline resistance.
Exam~le 6
Construction of Plasmid pNM608 and E. coli K12 RV308/
pNM608
A. Construction of the ~253 bp EcoRI-H aI Fragment of
Plasmid PHI7~4~1
About 5 ~g of plasmid pHI7~4~1 DNA, 10 ~1
(lOX) reaction buffer*, 80 ~1 water and 5 ~1 (5 units)
of HDaI restriction enzyme were incubated at 37C for
about 1 hour. The reaction was terminated by incubation
at 70~ for 5 minutes. After the mixture was cooled on
ice, about 12 ~1 of lM Tris-HCl, pH 7.2, 7 ~1 water and
1 ~1 (10 units) of EcoRI restriction enzyme were added.
The resultant mixture was incubated first at 37C for 1
hour and then at 70C for 5 minutes. Following cooling
on ice, the resultant DNA was extracted with each of
phenol and chloroform:isoamyl alcohol (24:1) and then
ethanol precipitated. The desired ~253 bp EcoRI-HpaI
fragment was conventionally separated by polyacrylamide
gel electrophoresis, recovered by electroelution and
ETOH precipitation and then dissolved in about 10 ~1 of
TE buffer and stored at 0C for future use.
~91`~
X-6125C-Canada -72-
Reaction buffer (lOX) for HPaI restriction enzymewas prepared with the following composition:
100 mM Tris HCl, pH 7
100 mM MgCl
60 mM ~-me~captoethanol
200 mM KCl
0 B. Construction of the ~34 bp H~aI~ I Fragment of
Plasmid PHI7~4~1
About 20 ~g of plasmid pHI7~4~1 DNA in 32 ~1
(5X) EcoRI reaction buffer*, 124 ~1 water and 4 ~1 EcoRI
restriction enzyme (20 units) were incubated at 37C for
about 1 hour. The reaction was terminated by incubation
at 70C for 5 minutes. The resulting 862 bp EcoRI
fragment was isolated by electrophoresis on a 6% poly-
acrylamide gel followed by electroelution and ethanol
precipitation. The fragment was then dissolved in about
100 ~1 of H~aI buffer containing 16 units of H~aI re-
striction enzyme. After 1.5 hours at 37C, 7.~ units of
~I restriction enzyme were added, incubation was
continued for an additional 1.5 hours and the resulting
~5 fragments conventionally separated on a 71~o polyacryl-
amide gel. The desired ~34 bp HPaI-_~gI fragment was
recovered by electroelution and ethanol precipitation
and was then dissolved in 5 ~1 of TE buffer.
Reaction buffer (SX) for EcoI restriction
enzyme was prepared with the following
composition:
P~91~8
X-6125C-Canada ~73~
250 mM NaCl
500 mM Tris-HCl, pH 7.2
25 mM MgCl
30 mM ~-me~captoethanol
C. EcoRI-ClaI Digestion of Plasmid pBR322
About 5 ~g of plasmid pBR322 DNA, 10 ~1 of
ClaI reaction mix*, 77.5 ~1 water and 7.5 ~1 (15 units)
of ClaI restriction enzyme were incubated at 37C for
about 1 hour. The reaction was terminated by incubation
at 70C for 5 minutes. After the mixture was cooled on
ice, about 12.S ~1 of lM Tris HCl, pH 7.2, 6.25 ~1 lM
NaCl, 2.5 ~1 O.lM MgC12 and 1 ~1 (10 units) of EcoRI
restriction enzyme were added. The resultant mixture
was incubated first at 37C for 1 hour and then at 70C
for S minutes. Following cooling on ice, the resultant
DNA was subjected to electrophoresis on a 1% agarose
gel. The large fragment was recovered by elution and
ethanol precipitated and then dissolved in about 20 ~1
of TE buffer and stored at 0C for future use.
Reaction mix (lOX) for ClaI restriction enzyme was
prepared with the following composition:5
100 mM Tris HCl, pH 7.4
100 mM MgCl
60 mM ~-me~captoethanol
l~1s~la
X-6125C-Canada _74_
D. Liaation and Transformation
About 0.2 ~g of the ~253 bp EcoRI-HPaI frag-
ment of Example 6A, 0.5 ~g of the HPaI- ~ I fragment of
Example 6B and 0.1 ~g of EcoRI-ClaI digest of Example 6C
were ligated and used to transform E. coli K12 RV308 in
substantial accordance with the teaching of Example 2D.
Some of the resultant transformants, as
conventionally shown by agarose gel electrophoresis
(Maniatis et al, 1982) and other tests, contained only
the desired ~4.6 kb plasmid. Such a transformant,
herein designated as E. coli K12 RV308/pNM608, was
selected, plated on TY agar containing appropriate
antibiotics and then cultured using conventional micro-
biological techniques. Plasmid pNM608 comprises and is
a preferred source of the ~287 bp EcoRI-ClaI (~
fragment of plasmid pHI7h4~1.
Example 7
Construction of Plasmid pCZ112 an- E. coli K12 RV308/
pCZ112
A. EcoRI-ClaI Digestion of Plasmid pNM608 and
Generation of the ~0.288 kb EcoRI~ I Fragment
(Same as the 0.288 kb EcoRI-TaqI Fragment of
Plasmid ~HI7~4~1)
About 5 ~g of plasmid pNM608 DNA in 50 ~1
Medium Salt buffer* were incubated with 10 units each of
E RI and ClaI restriction enzymes at 37C for about 1
~9~8
X-6125C-Canada -75~
hour. After the addition of 5 ~1 of 3M sodium acetate,
pH 7.0, the DNA was precipitated with 3 volumes of 100%
ethanol. The desired DNA digest was dissolved in 100 ~1
of TE buffer and stored at 0C for future use.
Medium Salt buffer was prepared with the following
composition:
50 mM NaCl
lQ0 mM Tris HCl, pH 7.5
6 mM MgCl
2 mM ~-me~captoethanol
B. Construction of the DNA Linker Sequence
5' CGACC ATG GAT GAT AAG TTT CCG GCT ATG TCT CTG
3' TGG TAC CTA CTA TTC AAA GGC CGA TAC AGA GAC
TCC GGC CTG TTT GCC AAC GCT GTGCT 3'
I ~ I I I I I I ~ t I I I I I I I I I I I I I t l I
AGG CCG GAC AAA CGG TTG CGA C 5'
The desired linker sequence is conventionally
synthesized by the modified phosphotriester method in
substantial accordance with the teaching of Itakura et
al, 1977 and Crea et al., 1978. The aforementioned
synthesis method is also specifically illustrated in
Example 1.
C. Liqation and Transformation
About 20 picomoles of the DNA linker of
Example 7B, 1 ~g of the pNM608 digest of Example 7A,
~9~
X-61 25C-Canada -76-
O.5 ~g of the plasmid pCZ101 ~10.2 kb BamHI-EcoRI frag-
ment (prepared in accordance with the teaching of
Example 2B except that EcoRI restriction enzyme and
appropriate buffer, rather than XbaI restriction enzyme
and buffer, are used), and 1 ~g of the plasmid pCZ101
~0.6 kb BamHI-HqiAI fragment of Example 3B are ligated
and the resultant plasmid used to transform E. coli K12
RV308 in substantial accordance with the teaching of
Example 2D.
Some of the resultant transformants, as
conventionally shown by agarose gel electrophoresis
(Maniatis et al., 1982) and other tests, contain only
the desired ~10.8 kb plasmid. Such a transformant,
herein designated as E. coli K12 RV308/pCZ112, is
selected, plated on TY agar containing appropriate
antibiotics and then cultured using conventional micro-
biological techniques. The resultant cells are shown,
by SDS gel electrophoresis, RIA and other tests, to
express the aforementioned MET-ASP-ASP-LYS-bGH deriva-
tive at high levels. Because plasmid pCZ112 contains athermoinducible runaway replicon, maximum expression of
the desired bGH derivative product occurs at culture
temperatures of about 37C.
~9i7~3
X-6125C-Canada -77-
ExamPle 8
Construction of Plasmid pATl and E. coli K12 RV308/-
DATl
The desired constructions are made in substan-
tial accordance with the teaching of Example 7 except
that the DNA linker sequence
5' CGACA ATG TTC CCA GCT ATG TCT CTA TCT GGT
''' "' ''' ''' ''' ''' ''' ''' '''
3' TGT TAC ~AG GGT CGA TAC AGA GAT AGA CCA
CTG TTT GCC AAC GCT GTGCT 3'
... ... ... ... ... .
GAC AAA CGG TTG CGA C S'
is substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned ~ynthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pAT1, are plated on TY agar con-
-
taining appropriate antibiotics and then conventionally
cultured for subsequent production and isolation of
plasmid pATl. The transformants are also shown, by SDS
gel electrophoresis, RIA and other tests, to express the
aforedefined MET-bGH derivative at high levels. Because
plasmid pATl contains a thermoinducible runaway replicon,
maximum expression of the desired bGH derivative product
occurs at culture temperatures of about 37C.
~91~8
X-6125C-Canada -78-
Exam~le 9
Construction of Plasmld pASPl and E. coli K12 RV308/-
PA-spl
The desired constructions are made in substan-
tial accordance with the teaching of Example 7 except
that the DNA seguence
5' CGACC ATG GAT TTT CCG GCT ATG TCT CTG TCC GGC CTG
-- -- -- -- .. ... ... ... ... ... ... ...
3' TGG TAC CTA AAA GGC CGA TAC AGC GAC AGG CCG GAC
TTT GCC AAC GCT GTGCT 3'
J AAA CGG TTG CGA C 5'
is substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pASPl are plated on TY agar con-
taining appropriate antibiotics and then conventionally
cultured for subsequent production and isolation of
plasmid pASPl. The transformants are also shown, by SDS
gel electrophoresis, RIA and other tests, to express the
aforedefined MET-ASP-bG~ derivative at high levels.
Because plasmid pASPl contains a thermoinducible runaway
replicon, maximum expression of the desired bGH deriva-
tive product occurs at culture temperatures of about
37C.
~;29~7i8 - -
X-6125C-Canada
Example lO
Construction of Plasmid pASP2 and E. coli K12 RV308/-
pASP2
The desired constructions are made in substan-
tial accordance with the teaching of Example 7 except
that the DNA seguence
5' CGATC ATG GAT TTT CCG GCT ATG TCT CTG TCC GGC CTG
... ... ... ... ... ... ... ... ... ... ... ...
3' TAG TAC CTA AAA GGC CGA TAC AGA GAC AGG CCG GAC
TTT GCC AAC GCT GTGCT 3'
,~ .- ... ... ... .
~ AAA CGG TTG CGA C 5'
is substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pASP2 are plated on TY agar con-
taining appropriate antibiotics and then conventionally
cultured for subseguent production and isolation of
plasmid pASPl. The transformants are also shown, by SDS
gel electrophoresis, RIA and other tests, to express the
aforedefined MET-ASP-bGH derivative at high levels.
Because plasmid pASP2 contains a thermoinducible runaway
replicon, maximum expression of the desired bGH deriva-
tive product occurs at culture temperatures of about
37C.
1~917~8
X-6125C-Canada -80-
ExamPle 11
Construction of Plasmid pAT2 and E. coli K12 RV308/-
pAT2
s
The desired constructions are made in substan-
tial accordance with the teaching of Example 3 except
that the DNA linker sequence
5' CTAGAGGGTATTAATA ATG TTT CCA GCT ATG TCT CTA TCT
............ ... ... ... ,.. .,. ... ... .. I
3' TCCCATAATTAT TAC AAA GGT CGA TAC AGA GAT AGA
G&T CTG TTT GCC AAC GCT GTGCT 3'
~c -. -- -- .. .. - .. - .
CCA GAC AAA CGG TTG CGA C 5'
is substituted for the linker sequence of Example 3C.
The above-specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pAT2, are plated on TY agar con-
taining appropriate antibiotics and then conventionally
cultured for subsequent production and isolation of
plasmid pAT2. The transformants are also shown, by SDS
gel electrophoresis, RIA and other tests, to express the
aforedefined MET-bGH derivative at high levels. Because
plasmid pAT2 contains a thermoinducible runaway replicon,
maximum expression of the desired bGH derivative product
occurs at culture temperatures of about 37C.
9~
X-6125C-Canada -81-
ExamDle 12
Construction of Plasmid pCZ154 and E. coli K12 RV308/-
pCZ154
The desired constructions were made in substan-
tial accordance with the teaching of Example 7 except
that the DNA linker se~uence
5' CGACC ATG GTT TTT CCG GCT ATG TCT CTG TCC GÇC CTG
... ... ....... ... ... ... ... ... ... ... ...
3' TGG TAC CAA AAA GGC CGA TAC AGA GAC AGG CCG GAC
TTT GCC AAC GCT GTGCT 3'
-- .. ,,, , " ,
AAA CGG TTG CGA C 5'
was substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pCZ154, were plated on TY agar
containing appropriate antibiotics and then convention-
ally cultured for subsequent production and isolation of
plasmid pCZ154. The transformants were also shown, by
SDS gel electrophoresis, RIA and other tests, to express
the aforedefined ~ET-VAL-bGH derivative at high levels.
Because plasmid pCZ154 contains a thermoinducible
runaway rep~icon, maximum expression of the desired bGH
derivative product occurs at culture temperatures of
about 37C.
~.~..,9~
X-6125C-Canada -82-
Exam~le 13
Construction of Plasmid pCZ155 and E. coli K}2 RV308/-
pCZ155
The desired constructions were made in substan-
tial accordance with the teaching of Example 7 except
that the DNA linker sequence
~' CGACC ATG GCT TTT CCG GCT ATG TCT CTG TCC GTC CTG
... .-- .. ... ..~ ... ... ... ... ..~ ... I.I
3' TGG TAC CGA AAA GGC CGA TAC AGA GAC AGG CAG GAC
TTT GCC AAC GCT GTGCT 3'
AAA CGG TTG CGA C 5'
was substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al., 1977 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli K12 RV308/pCZ155, were plated on TY agar
containing appropriate antibiotics and then convention-
ally cultured for subsequent production and isolation of
plasmid pCZ155. The transformants were also shown, by
SDS gel electrophoresis, RIA and other tests, to express
the aforedefined MET-ALA-PHE-PRO-ALA-MET-SER-LEU-SER-
VAL-b'GH derivative at high levels. Because plasmid
pCZ155 contains a thermoinducible runaway replicon,
maximum expression of the desired bGH derivative product
occurs at culture temperatures of about 37~C.
~917~3
X-6125C-Canada -83-
Example 14
Construction of Plasmid pCZ156 and E. coli Kl2 RV308/-
pCZ156
The desired constructions were made in substan-
tial accordance with the teaching of Example 7 except
that the DNA linker sequence
5' CGATA ATG GAT TTT CCG GCT ATG TCT CTG TCC GGC CTG
3' TAT TAC CTA AAA GGC CGA TAC AGA GAC AGG CCG GAC
TTT GCC AAC GCT GTGCT 3'
-. ,,, , " ,,, ,
AAA CGG TTG CGA C 5'
was substituted for the linker sequence of Example 7.
The above specified linker sequence can be constructed
in substantial accordance with the conventional proce-
dure of Itakura et al,, lg77 and Crea et al., 1978. The
aforementioned synthesis method is also specifically
illustrated in Example 1.
The desired transformants, herein designated
as E. coli Kl2 RV308/pCZl56, were plated on TY agar
containing appropriate antibiotics and then convention-
ally cultured for subseguent production and isolation of
plasmid pCZl56. The transformants were also shown, by
SDS gel electrophoresis, RIA and other tests, to express
the aforedefined MET-ASP-bGH derivative at high levels.
Because plasmid pCZ156 contains a thermoinducible
runaway replicon, maximum expression of the desired bGH
derivative product occurs at culture temperatures of
about 37C.
~2~917~
X-6125C-Canada -84-
ExamPle }5
Construction of Plasmid pCZ103 and E. coli K12 RV308/pCZ103
Ten ~g of plasmid pCZ101 were dissolved in
10 ~1 10X BstEII reaction buffer (1.5 M NaCl; 60 mM
Tris-HCl, pH=7.9; 60 mM MgC12; 60 mM 2-mercaptoethanol
and 1 mg/ml BSA), 2 ~ 20 units) restriction enzyme
BstEII, and 88 ~1 of H2O, and the resulting reaction was
incubated at.60C for two hours. After phenol and
chloroform extractions, several ethanol precipitations
of the DNA were done from a 0.25 M NaOAc solution.
About 0.1 ~g of the BstEII-digested plasmid
pCZ101 DNA was suspended in 100 ~1 of ligase buffer.
After adding 100 units of T4 DNA ligase and incubating
at 16C for 2 hours, the ligated DNA was used to conven-
tionally transform E. coli K12 RV308. E. coli K12
RV308/pCZ103 transformants were selected on kanamycin
and identified by restriction enzyme analysis of their
plasmid DNA. Plasmid pCZ103 was isolated from the
transformants in substantial accordance with the pro-
cedure of Example 2A.
~ 29~7~8
X-6125C-Canada -85-
Exam~le 16
Construction of Plasmld pCZ103-Derived Vectors For
Ex~ressinq Bovine Growth Hormone Derivatives
Since the BstEII deletion done to construct
plasmid pCZ103 does not affect the bGH protein-coding
sequence, the plasmid pCZ103-derived plasmids of the
present invention express the same bGH derivative as
their plasmid pCZlOl~derived counterparts.
Since the pCZ103-derived plasmids of the
present invention were constructed in substantial
accordance with the teaching of the Examples in which
their pCZ101-derived counterparts were constructed,
Table 3 is presented to briefly summarize the con-
struction of the pCZ103-derived plasmids. Table 3
lists the pCZ103-derived plasmid, the corresponding
Example teaching the construction of the pCZ101-
derived counterpart, and the size of the only dif-
ferent DNA fragment involved in the construction.
X-6125c-Canada -86- ~L~191~18
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t917~8
X-6125C-Canada -87-
The pCZ103-derived plasmids were used to con-
ventionally transform E. coli K12 RV308. The resultant
transformants express the aforementioned bGH derivatives.
Because the pCZ103-derived plasmids contain a thermo-
inducible runaway replicon, maximum expression occursat culture temperatures of about 37C.
