Note: Descriptions are shown in the official language in which they were submitted.
4~
"THE ~ANUFACTURE AND EXPRESSION OF GENES FOR
UROGASTRONE AND POLYPEPTIDE ANALOGS THEREOF"
BACKGROUND
The present invention relates generally
to the manipulation of genetic materials and, more
particularly, to the manufacture of specific DNA
sequences useful in recombinant procedures to secure
the production of urogastrone and polypeptide analogs
thereof.
Incorporated by reference herein for the
purpose of providing information pertinent to the
prior art with respect to recombinant DNA techniques
is co-owned, co-pending Canadian Patent Application
Serial No. 427,371, filed May 4, 1983, inventor
Yitzhak Stabinsky, entitled "Manufacture and Expression
of Structural Genes".
A component in human urine which inhibits
gastric acid secretion was first described by Gray
in 1939 [Gray, et al., Science, 89, 489 (1939)].
This component, named "urogastrone" was completely
sequenced and its structure was published in 1975
~2~
-- 2 --
[N. Gregory, Nature _(London~, 257, 325 (1975)]. Earlier,
the isol~tion and characterization of a factor from
mouse salivary glands which promotes the growth of
epidermal tissue had been published lCohen, J. Biol.
Chem., 237, 1555 (1962)]. This compound was called
"epidermal growth ~actor". When the amino acid composi-
tion of epidermal growth factor was compared with
that of urogastrone, it was found that the two peptides
were closely related. It is now known that these
compounds, mouse and human epidermal growth factor-
urogastrone (EGF-URO), are examples of a large class
of "growth factors" and ar~ widespread in animals
and man. ~~
EGF-URO like the other growth ~actors such
as insulin, nerve growth factor, the insulin-like
growth factors, and the like, is synthesized in mammals
as part of a larger "pro-peptide" molecule from which
it is cleaved by specific proteases to liberate the
active form of the protein rFrey, et al.~ Proc. Nat.
Acad. Sci., 76, 6294 (1979)]. When cleaved from its
pro-peptide, EGF-URO, in both the mouse and in man,
is composed of 53 amino acids. Further processing
in the body also gives rise to a 51 amino acid-contain-
ing form which lacks the two amino acid residues at
the carboxvL terminus of the peptide. The 53 and
51 amino acid forms of the peptide are called beta-
and gamma- EGF-URO, respectively. Both forms have
shown high activity as inhibitors of gastric acid
secretion and as stimulators of growth of epidermoid
30 tissue. High gastric secretion inhibitory activi-
ties have also been reported for the 46 and 47 amino
acid products of selective enzymatic degradation.
[See, U.S. Patent Nos. 4,032,633 and 4,035,485.]
Receptors for EGF-URO have been found in
various tissues of the human, mouse, rat, chicken,
rabbit, cow; monkey, dog, cat, mink and hamster [Adamson,
et al., Mol. Cell. Biochem., 34, 129 (1981)]. Work
. .
~æ~ 73~ i
- 3 -
done with mouse and human EGF-URO has shown that they
have identical activities in both species, the best
documented of which are the abilities to virtually
stop gastric acid secretion and to cause prolifera- !
tion of epidermal and other epithelial tissues. ESee,
e.g, Starkey, et al., Science, 189, pp. 800-803 (1974)
and Carpenter, Birth Defects: Original Article Series,
6, pp. 61-72 (1980)].
Despite its significant biological activities,
little has been done to explore the full clinical
potential of urogastrone and synthetic analogs thereof.
This is due in large part to lack of large quantities
of the substance. EGF-URO is presently isolated in
small quantities by purification from mouse salivary
glands or by a complex purification from human urine
[Hollenberg, Vitamins and Hormonest 37, 69 (1979);
Gregory et al., U.S. Patent No. 3,883,497].
The polypeptide substance is too large to
be readily synthesized by the well-known Merrifield
procedure. Recombinant DNA techni~ues for the manufac~
ture J cloning and expression of a structural gene
for urogastrone and genes for polypeptide analogs
which differ therefrom in terms of the identity and/or
location of one or more amino acids have not been
brought to bear on this problem.
BRIEF SUMMARY
Provided by the present invention is a manu-
factured gene capable of directing synthesis in a
selected host microorganism of urogastrone. In a
preferred form of manufactured gene, the base sequence
includes one or more ~odons selected from among alterna-
tive codons specifying the same amino acid on the
basis of preferential expression characteristics of
the codon in a projected host microorganism, e.g.,
E. coli. Other preferred forms of manufactured genes
~.;~,~73g
-- 4
include those wherein: (1) a base codon specifying
additional amino acid in the polypeptide synthesized
which facilitates direct expression in E. coli organisms
(e.g., an initial Met residue) and/or (2) base codons
specifying urogastrone are preceded and/or followed
by a sequence of bases comprising a portion of a base
sequence which provides for restriction endonuclease
cleavage of a DNA sequence (e.g., a BclI or Ba~HI
site) and consequently facilitates formation of expres-
sion vectors.
~lso provided by the present invention are:
(1) a manufactured gene capable of directing the synthe-
sis in a selected host microorganism of a urogastrone
polypeptide analogs which differ from urogastrone
in terms of the identity and/or location of one or
more amino acids (e.g., [Asp25] urogastrone and [Pro52,
Pro 53] urogastrone); and (2) a fusion gene comprising
a manufactured gene according to the invention fused
to a second gene capable of directing synthesis of
a second gene capable of directing synthesis of a
second polypeptide (e.g., ~-lactamase and ~-galacto-
sidase) in a manner permitting the synthesis of a
fused polypeptide including urogastrone polypeptide
or a urogastrone analog.
In practice of the invention to generate
polypeptide products, DNA sequences including manufac-
tured genes are inserted into a viral or circular
plasmid DNA vector to form a hybrid vector and the
hybrid vectors are employed to transform host micro-
organisms such as bacteria (e.g., E. coli) or yeast
cells. The transformed microorganisms are thereafter
grown under appropriate nutrient conditions and express
the polypeptide products of the invention.
Novel DNA sequences of the invention are
preferably synthesized from nucleotide bases according
to the methods disclosed in the aforementioned co-owned,
concurrently-filed Canadian Patent Application Serial
No. 427,371, inventor Yitzhak Stabinsky, entitled
`` 3~ ~ 1
- 5 -
"Manufacture and Expression of Structural Genes".
Briefly summarized, the general method comprises the
steps of:
(1) preparing two or more different, linear,
duplex DN~ strands, each duplex strand including a
double stranded region of 12 or more selected complemen~
tary base pairs and further including a top single
stranded terminal sequence of from 3 to 7 selected
bases at one end of the strand and/or a bottom single
stranded terminal sequence of from 3 to 7 selected
bases at the other end of the strand, each single
stranded terminal sequence of each duplex DNA strand
comprising the entire base complement of at most one
single stranded terminal sequence of any other duplex
DNA strand prepared; and
(2) annealing each duplex DNA strand prepared
in step (13 to one or two different duple~ strands
prepared in step (1) having a complementary single
stranded terminal sequence, thereby to form a single
continuous double stranded DNA sequence which has
a duplex region of at least 27 selected base pairs
including at least three base pairs formed by complemen-
tary association of single stranded terminal sequences
of duplex DNA strands prepared in step (1) and which
has from 0 to 2 single stranded top or bottom terminal
regions of from 3 to 7 bases.
In the preferred general process of manufac-
ture, at least three different duplex DNA strands
are prepared in step (1) and all strands so prepared
3~ are annealed concurrently in a single annealing reaction
mixtu~e to form a single continuous double stranded
DNA sequence which has a duplex region of at least
42 selected base pairs including at least two non-
adjacent sets of 3 or more base pairs formed by comple
mentary association of single stranded terminal sequen-
ces o~ duplex strands prepared in step (1).
~ 4q3gl
-- 6 --
The duplex DNA strand preparation step (1)
of the DNA sequence manufacturing process noted above
preferably comprises the steps of:
(a) constructing first and second linear
deoxyoligonucleotide segments having 15 or more bases
in a selected linear sequence, the linear sequence
of bases of the second segment comprising the total
complement of the sequence of bases of the first segment
except that at least one end of the second segment
shall either include an additional linear sequence
of from 3 to 7 selected base~ beyond those fully comple-
menting the first segment~ or shall lack a linear
- sequence of from 3 to 7 bases complementary to a ter-
minal sequence of the first segment, provided, however,
that the second segment shall not have an additional
sequence of bases or be lacking a sequence of bases
at both of its ends; and,
(b) combining the first and second segments
under conditions conducive to complementary association
between segments to form a linear, duplex DNA strand.
The sequence of bases in the double stranded
DNA subunit sequences formed preferably includes one
or more triplet codons selected from among alternative
codons specifyir.g the same amino acid on the basis
o~ preferential expression characteristics of the
codon in a projected host microorganism, such as yeast
cells or bacteria, especially E. coli bacteria.
Other aspects and advantages of the present
invention will be apparent upon consideration of the
following detailed description thereof.
DETAILED DESCRIPTION
As employed herein, the term "manufactured"
as applied to a DNA sequence or gene shall designate
a product either totally chemically synthesized by
~Z31~73~i;
-- 7 --
assembly of nucleotide bases or derived from the bio-
logical replication of a product thus chemically synthe-
sized. As such, the term is exclusive of products
"synthesized" by cDNA methods or genomic cloning method-
ologies which involve starting materials which areinitially of biological origin.
The following abbreviations shall be employed
herein to designate amino acids: Alanine, Ala; Arginine,
Arg; Asparagine, Asn; Aspartic acid, Asp; Cysteine,
Cys; Glutamine, Gln; Glutamic acid, Glu; Glycine,
,~. Gly; Histidine, His; Isoleucine, Ile; Leucine, Leu;
Lysine, Lys; Methionine, Met; Phenylalanine, Phe;
Proline, Pro; Serine, Ser; Threonine, Thr; Tryptophanr
Trp; Tyrosine, Tyr; Valinel Val. The following abbrevia-
tions shall be employed for nucleotide bases: A for
adenine; G for guanine; T for thymine; U for uracil;
and C for cytosine~
For ease of understanding of the present
invention, Table I below provides a tabular correlation
between the 64 alternate triplet nucleotide base codons
of DNA and the 20 amino acids and transcription termina-
tion ("stop") function specified thereby.
-- 8 --
TABLE I
POSITION SECOND POSITION POSITION
T C A G
__
Phe Ser Tyr Cys T
Phe Ser Tyr Cys C
T Leu Ser Stop Stop A
Leu Ser Stop Trp G
.
,~- Leu Pro His Arg T
Leu Pro His Arg C
~ Leu Pro Gln Arg A
Leu Pro Gln Arg G
. . _
Ile Thr Asn Ser T
Ile Thr Asn Ser - C
Ile Thr Lys Arg A
Met Thr Lys Arg ~ G
Val Ala Asp Gly T
Val Ala Asp Gly C
G Val Ala Glu Gly A
Val Ala Glu Gly G
- - - - ----- -
The following example illustrates a preferred
general procedure for preparation of deoxyoligonucleo-
tides for use in the manufacture of DNA sequencesof the invention.
EXAMPLE 1
Oligonucleotide fragments were synthesized
using a four-step procedure and several intermediate
washes. Polymer bound dimethoxytrityl protected nucleo-
- 9 -
side in a sintered glass funnel was first stripped
of its 5'-protecting group (dimethoxytrityl) using
3% trichloroacetic acid in dichloromethane for 1 1/2
minutes. The polymer was then washed with methanol,
tetrahydrofuran and acetonitrileO The washed polymer
was then rinsed with dry acetonitrile, placed under
argon and then treated in the condensation step as
follows. 0.5 ml of a solution of lO mg tetrazole
in acetonitile was added to the reaction vessel contain-
ing polymer. Then 0 5 ml of 30 mg protected nucleosidephosphoramidite in acetonitrile was added. This
reaction was agitated and allowed to react for 2 minutes.
The reactants were then removed by suction and the
polymer rinsed with acetonitrile. This was followed
by the oxidation step wherein l ml of a solution contain-
ing 0.1 ~olar I2 in 2-6-lutidine/H20/THF, 1:2:2, was
reacted with the polymer bound oligonucleotide chain
for 2 minutes. Following a THF rinse capping was
done using a solution of dimethylaminopyridine (6.5 g
in lO0 ml THF3 and acetic anhydride in the proportion
4:1 for 2 minutes. This was followed by a methanol
rinse and a THF rinse. Then the cycle began a~ain
with a trichloroacetic acid in C~2Cl2 treatment.
The cycle was repeated until the desired oligonucleotide
sequence was obtained.
The final oligonucleotide chain was treated
with thiophenol dioxane, triethylamine 1:2:2, for
45 minutes at room temperature. Then, after rinsing
with dioxane, methanol and diethylether, the oligonucleo-
tide was cleaved from the polymer with concentrated
ammonia at room temperature. After decanting the
solution from the polymer, the concentrated ammonia
solution was heated at 60C for 16 hours in a sealed
tube.
Each oligonucleotide solution was then extrac-
ted four times with l-butanol. The solution was loaded
~L~ 3~1
-- 10 --
into a 20~ polyacrylamide 7 molar urea electrophoresis
gel and, after running, the appropriate product band
was isolat~d
The following example illustrates the prepara
tion of a DNA sequence which comprises a gene coding
for [Met 1] urogastrone and which includes terminal
base sequences facilitative of insertion of the sequence
into DNA plasmid restriction sites.
EXAMPLE 2
The following deoxyoligonucleotides were
synthesized according to the procedures of Example 1.
1. 5'-GG TGG GAA CTG CGT TAA TAG-
2. 5'-CAG TAC CGT GAT CTG AAA T
3. 5 '-GT TAC ATC GGT GAA CGT TGC
4. 5'-GCT TGC AAC TGC GTA GTT G
5. 5'-GTA ACC AAC TAC GCA GTT G
6. 5'-TACTG GCA ACG TTC ACC GAT
7. 5'-CCA CCA TTT CAG ATC ACGG
8. 5'-GATC CTA TTA ACG CAG TTC
9. 5'-G TAT ATC GAA GCT CTG GAC AAA TAC
10. 5'-AT TGT CTG CAC GAC GGT GTT TGC AT
25 11. 5'-AA TGC CCG CTG TCC CAC GAC GGT T
12. 5'-G ATC ACA ATC AAC TCT GAT TCC G
13. 5'-GCA TTC GGA ATC AGA GTT CAT TGT
14. 5'-ACA ATA ACC TGC GTG GGA CAG CGG
15. 5'-AT ATA CAT GCA AAC ACC GTC GTG CAG
16. 5'-CA AGC GTA TTT TGC CAG AGC TTC G
The oligonucleotide sequences purified by
polyacrylamide gel electrophoresis were phosphorylated
at the 5' ends using ATP and T4 polynucleotide kinase
in a standard reaction using one nanomole of DNA,
a two fold excess of ATP and 1 unit of T4 kinase in
S~ 3~3
20 ~1 of buffer made with 50 mM hydroxyethylpiperazine
ethane s~lfonic acid, 10 mM MgC12, 10 mM dithiothreitol,
pH 7.6. After reaction, the kinase was destroyed
by boiling for 5 minutes. These phosphorylated oligo-
nucleotides in the buffer were then used directlyfor ligation. These sequences are shown in Table 1.
The oligonucleotides in 20 ~1 standard buffer
were combined to form short duplexes. Each duplex
was formed by combining two complementary sequences
in equimolar amounts, boiling the mixture, then slow
cooling over a 1/2 hour period to room temperature.
In this way, the duplexes in Table II were formed.
TABLE II
(12~
G ATC ACA ATG AAC TCT GAT TCC G
TGT TAC TTG AGA CTA AGG CTT ACG
(13)
(11)
AA TGC CCG CTG TCC CAC GAC GGT T
GGC GAC AGG GTG CTG CCA ATA ACA
(14)
(~0)
AT TGT CTG CAC GAC GGT GTT TGC AT
GAC GTG CTG CCA CAA ACG TAC ATA TA
(15)
(9)
G TAT ATC GAA GCT CTG GAC AAA TAC
G CTT CGA GAC CTG TTT ATG CGA AC
(16)
(4)
GCT TGC AAC TGC GTA GTT G
3 0 G TTG ACG CAT CAA CCA ATG
(5)
(3)
GT TAC ATC GGT GAA CGT TGC
TAG CCA CTT GCA ACG GTC AT
(6)
~4~73~
- 12 -
(2)
CAG TAC CGT GAT CTG AAA T
G GCA CTA GAC TTT ACC ACC
t7)
(1)
G~ TGG GAA CTG CGT TAA TAG
CTT GAC GCA ATT ATC CTA G
(8)
These 8 duplexes were combined sequentially,
annealing each set of duplexes at 37C for 5 minutes
until the final structural gene was in a single tube
ready for ligationO The oligonucleotide mixture was
then made 150 ~molar in ATP and treated wi~h 84 ~nits
of T4DNA ligase for 16 hours at 4C. The fully ligated
structural gene was then purified by polyacrylamide
gel electrophoresis. The final structural gene wi~h
appropriate restriction sites is shown in Table III.
This was a 175 base pair duplex having Bcl I restriction
site at the amino terminal end and a Bam HI site at
the carboxy terminal end.
`!
739~
- 13 -
TABLE III
-1 1 2 3 4 5 6 7 8 9
Bcl I -Met-Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-
G ATC ACA ATG AAC TCT GAT TCC GAA TGC CCG CTG TCC-
5TGT TAC TTG AGA CTA AGG CTT ACG GGC GAC AGG-
11 12 13 14 15 16 17 1~ 19 20 21
His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gly-Val Cys-Met-
CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-
GTG CTG CCA ATA ACA GAC GTG CTG CC~ CAA ACG TAC-
22 23 24 25 26 27 28 29 30 31 32 33
Tyr-I le-Glu-Ala Leu-Asp-Lys-Tyr-Ala-Cys-Asn-Cys-
TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-
ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-
1534 35 36 37 38 39 40 41 42 43 44 45
Val-Val-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-Try-Arg-
GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-
CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-
46 47 4~ 49 50 51 52 53
20Asp-Leu-Lys-Trp-Trp-Glu-Leu-Arg-Stop Stop-
GAT CTG AAA TGG TGG GAA CTG CGT TAA TAG-
CTA GAC TTT ACC ACC CTT GAC GCA ATT ATC CTA G-
Bam HI
Mutant genes coding for polypeptide sequences
different from the natural sequence were also prepared.
This was done by changing selected segments and repeat-
ing the ligation step to obtain the new genes. By
altering segments 9 and 16, the alanine at residue
25 was changed to aspartic acid. The codon modification
was from GCT to GAT. This changes a neutral amino
acid residue to an acidic residue and may produce
a peptide with novel characteristics. Another mutant
gene was prepared by changing codons in segments 1
and 8. Specifically, codons for the Leu52-Arg53 resi-
dues (5'-CTG CGT-3') were replaced by those coding
for Pro5 , Pro53 (5'-CCG CCA-3'). This gene should
^~i - ~7;3~
- 14 -
code for a peptide resistant to enzyme degradation,
but stil~ retaining its other desirable properties.
The following example relates to cloning
of the [Met 1] urogastrone gene prepared in Example ~.
EXAMPLE 3
The 175 base pair HEGF-URO synthetic gene
was inserted into the E. coli cloning vector pBR325
using the restriction endonuclease sites BclI and
BamHl. Because the restriction sites have the same
cohesive termini J the gene was insertable in both
orientations. However, because both restriction sites
are destroyed by insertion of the gene in the incorrect
orientation, only those clones which contained the
gene in the correct orientation were excisable with
BclI and BamHl. Those clones with the gene in the
correct orientation were characterized by polyacrylamide
gel electrophoresis to verify the estimated molecular
weight for the urogastrone structural geneO
To further characterize the cloned synthetic
DNA segment, the 175 base pair fragment was excised
from the chimeric pBR325 plasmid (pHEGFl) and inserted
into single-strand bacteriophage M13mp8 relicative
form DNA at its BamHl site. Clones with the inserted
D~A in a defined orientation were isolated and character-
ized by polyacrylamide gel eletrophoresis. Single-
strand phage ~or one orientation were isolated and
the DNA sequence for the urogastrone structural gene
has been determined using the Sanger Dideoxy sequencing
technique,
Restriction endonuclease BclI cleaves plasmid
p~EGFl at its unique BclI site lying 7 nucleotides
5' to the translation initiation codon of the uro~as-
trone gene. Approximately 750 nucleotides 5' to thisrestriction site is a unique restriction endonuclease
739
-- 15 -- .
EcoRl site. Cleavage of pHEGF1 with EcoRl and BclI
permitted the insertion of a A PR promoter under control
of lac repressor between these restriction sites by
ln vitro recombination to create pHEGF5. Cloning
the A PR promoter using this approach insured correct
orientation of the A PR-lac promoter-operator relative
to the urogastrone structural gene~ The A PR promoter
under lac control used for this construction was an
84 base pair EcoRl BamHl excisable synthetically derived
DNA segment in E. coli cloning vector ~BR322. The
,~ BamHl restriction site of the promoter lies one nucleo-
tide 3' to the Shine-Dalgarno sequence. Consequently,
fusion of the A PR lac promoter with the urogastrone
structural gene at their BamHl - BclI cohesive termini
junction creates a ribosome binding site with eight
nucleotides between the Shine-Dalgarno sequence and
the HEGF-URO translation initiation codon. This is
close to optimal relative positioning for these two
elements. The insertion of the A PR promoter in the
correct orientation has been verified by restriction
enzyme analysis and molecular weight sizing using
polyacrylamide gel electrophoresis.
The A PR-lac-HEGF 259 base pair segment
was excised from pHEGF5 using Eco~:L and BamHl restric-
tion endonuclease digestion. This fragment was inserte3into EcoRl-BamHl digested pBR322 to construct pHEGF10.
This construction was performed because pBR322- expressed
proteins are more easily analyzed in a maxicell system
than pBR325-expressed proteins. In addition, pBR322
is a higher copy number plasmid than pBR325, conse-
quently urogastrone should be expressed in greater
amounts in pBR322. The insertion of the-A PR-lac-
HEGF DNA segmen~ has been verified by restriction
enzyme analysis and polyacrylamide gel electrophoresis.
E. coli containing pHEGF5 and pHEGF10 are
being examined for expression of urogastrone polypeptide
~2~L~'73~
- 16 -
products using the maxicell system. Polypeptide products
can be characterized using immunoprecipitation and/or
radioimmunoassay techniques with rabbit IgG to mouse
EGF.
The following example illustrates the prepara-
tion of a llNA seguence which comprises a gene coding
for [Met 1] urogastrone and which includes terminal
base seguences facilitative of insertion of the sequence
into DNA plasmid restriction sites as well as internal
base sequences facilitative of disassembly and reconstruc-
tion of selected portions of the gene.
EXAMPLE 4
The following deoxyoligonucleotides were
synthesized according to the general procedures of
Example 1.
1. GATCCAA ATG AAC TCT GAT TCC GAA T
2. GC CCG CTG TCT CAT GAC GGT TAC T
3. GC CTG CAT GAT GGC GTA TGC ATG TA
4. C ATC GAA GCT CTG GAC AAA TAC GCA
5. TGC AAC TGT GTT GTA GGT TAC ATC G
6. GC GAA CGT TGC CAG TAT CGC GAC CT
7. G AAA TGG TGG GAA CTG CGT TAA TAG
8. GG GCA TTC GGA ATC AGA GTT CAT TTG
9. CAG GCA GTA ACC GTC ATG AGA CAG C
10. C GAT GTA CAT GCA TAC GCC ATC ATG
11. TT GCA TGC GTA TTT GTC CAG AGC TT
30 12. TTC GCC GAT GTA ACC TAC AAC ACA G
13. A TTT CAG GTC GCG ATA CTG GCA ACG
14. TCGA CTA TTA ACG CAG TTC CCA CC
The oligonucleotides were combined to form
duplexes and sequentially annealed as in Example 2
to yield the structural gene set out in Table IV,
~73~
- 17 -
having bases forming the "sticky end" of a BamHI restric-
tion sité (prior to the polypeptide coding region)
and a SalI site (following the transcription termination
codons). While the codon usage generally invo.ved
selection based on projected use of an E.coli bacterial
expression system, the codons employed in this gene also
resulted in generation of internal recognition sites
for cleavage by, e.g., HinfI (5'-GATTC-3'), SphI (5'-
GCATGC-3') and NruI ~5'-TCGCGA-5').
. . .
.~ ~4~3~
-- 18 --
cn ~ ~ 1 ~ E~ ~:
,1 ~ E~ ~: o ~ ~ ~ t~
a: ~ C~ ~o -t
_l ~ ~ ~~r _l ~ E~
C~ V V
I` ~. E~ ~~ ~ C~ V
,~ ~ V ~~ V ~ V
ID ~ E~ ~o~ ~ U
`~ ,1 E~ ~:
:~1 ~ ~ ~ ~ E~
1;~) V ~-- h C,) V
E~ 1t~l ~ É~ ~
u~ C~ V~ ~ E~
_~ ~ ~
C~ ~ ~ ~ C~ V
CJ ~u~ ~ ~ E~
~ E~~ ~ E~
E~ ~::~
E~ ~ ~ E~
V C~~ ~ u ~ E~ ~ u~
C~ ~ ~ C~ C~ ~C
.~ ~, ~ C~ c ~ ~ ~q E~ ~ ~ -~ V
.~ lQ ~ E~~ :~t ~ C~
V W V E~ ~: u~ E~
o u~ E~ IcC~ ~: C,) U Q. ~! E~
r~rl ~ E.~~ ~n ~ E~ ~ ~ ~ E~
O ~ ~ ~ E~ S E~
`I E.~ ~C .~ ~.q_~) ~ ~ ~ C~ E~ ~
a c,~ ~~ :~ ~ U c~u) ~ ~ C)
H U~ E~ a O E~ ~:~: C~ V
~11~ ~ E~ ~ ~ g ~ ~ ~ ," ~
a1 ~ ~ W ~ C~ ~ ~ O ~_1
r~ V U-- a~ ~ E~ u~ ~ E~
tll U C~ E~ E~ ~ V V V
~ C) vco rn ~ E~o ~, V O
D >1 _V ~ ~ ~ ~ E~ u~
V E~ ~1 ~ ~ E~ r
~ .¢ E~r~ ~ ~ ~ V
u~ ~ ~ ~ ~n ~ E~~r h C~
~ C.~ C ~~ V ~ E~ h
er ~1) _~ C!7 1-1 N a~ E~ ~ co ~ E~
~n E~ ~ ~ ~ C~ C~ ~ E~
E~ ~: ~u~ a e r E~ .¢ ~_
E.:l . ~ .~ t~ V ~ ~ ~
SJ E~ ~:er 3 ~ u~ ~4 V V
V ~ .~ ~ E~~r ~,q ~ ~
t~ E.~ ~ c ~ C~ U C~ ~: ~ H
c~ ~ ~ a c~ vu~ ~ vl~ ,
.~ m ~ E~~ .~ ~ ~er ~ '~1~ ~
f~l ~ 1~ H ~1: E~~ C)IV Z
JJ V U ~ ~ _~ Ver ~ E~ ~:
.~ a) .~ E~ ~:C ~ :~ ~ ~ :~, ~: E.
~ E~ E~ E.~ ~:E.~ E~
~E~ ,~ C~V ~ ~ VC~' ~
~: E~~ aJ E~ ~:Cer ,~ I'G E~
V ~ ~ ~1: E~r~ V V
E~ E~ o u~ V V~ ~Qu ~ U V
~ ~ ~ ~ V Ver :~.C~ o
m _v v E.~ ,3C~E~ ~:
739
- 19 - . ,
The assembled sequence of Table IV was ampli-
fied by insertion into a BamHI/SalI cleaved Ml3 mp9
vector and then ligated to an EcoRI/BamHI DNA "linker"
constructed with an internal XbaI recognition site,
as set out in Table V.
TABLE V
EcoRI XbaI BamHI
GAA TTC TE~ A ATG AAG AAA TAT TG
AGA T~k TAC TTC TTT ATA ACC TAG
Thus provided with an adenosine-rich series
of bases prior to the urogastrone polypeptide-specifying
sequences, the construction was excised from an ampli-
fication plasmid with XbaI and SalI and inserted into
a pBR322-derived plasmid (pINT-y-TXb4) at a manufac-
tured XbaI site following the trp promoter/regulator
DNA seql~ence. The resulting vector r designated pAD~25,
was employ~d as an expression vector in a E. coli
host to generate a polypeptide including a "pro" sequence
of 8 amino acids, as set out below, prior to urogastrone
polypeptide:
-a -7 -6 -5 -4 -3 -2 -1
NH2-Met-Lys-Lys-Tyr-Trp-Ile-Gln-Met-[Urogastrone~.
The microbially expressed polypeptide dis-
played the biological activity of naturally-occurring
human urogastrone. The levels of expression of the
product as determined by bioassay procedures discussed
infra were on the order of fifteen micrograms per
O.D. liter.
``` ~.æ~4~73~
- 20 -
The following example relates to presently
preferre~ procedures for enhancing the levels of expres-
sion of products of the invention.
EXAMPLE 5
Plasmid pADH25 was treated with EcoRI and
SalI to isolate the entire urogastrone protein codin~
region (including the DNA sequence coding for the
eight residue "pro" sequence) and the entire trp pro-
moter/regulator DNA sequence. The EcoRI/SalI fragment
was inserted in a DNA vector containing a temperature
sensitive mutation in the copy control region. After
transformation with the vector, the host cells normally
contain a low copy number of the vector when grown
at temperatures of less than 34C. The plasmid copy
number increases 50-fold (i.e., "runs away") within
the host cell upon elevation of culture temperature
above 34C. Growth at 37C or above wiLl ordinarily
be lethal to the transformed host cells.
The new plasmid containing the above-noted
EcoRI/SalI insert from pADH25 was designated pADH59.
The plasmid was employed to transform E. coli K-12
JM103 cells ~Bethesda Research Labs.) and samples
~5 of the strain harboring pADH59 have been deposited
under contract with the American Type Culture Collec-
tion, Rockville Maryland as A.T.C.C. 393335.
The level of expression of urogastrone analog
product by A.T.C.C 393335 was on the order of fifty
milligrams per O.D. liter as determined by SDS-PAGE.
The following example relates to a bioassay
employed to assess the levels of microbial expression
of polypeptides of the present invention.
7~
- 21 -
EXAMPLE 6
A radioreceptor bioassay was developed to
assay for biological activity of microbially-expressed
products of the invention and was generally patterned
on the procedures of Fabricant, et al., P.N.A.S._~.S.A.,
74, pp. 565-569 tl977). Briefly put, the assay is
a competitive receptor binding assay wherein the amount
of urogastrone activity in an unknown sample is deter-
mined by the ability to displace radiolabelled urogastrone from bound association with cells in culture.
More specifically, cells of human epidermoid carcinoma
cell line A-431 are grown in culture and incubated
with fixed quantities of I125-labelled urogastrone
(Collaborative Research, Boston, MA.) which binds
to specific URO-EGF receptors on the cell surface.
The cells are w~shed to remove excess, unbound labelled
materials. Microbial cells transformed for production
of urogastrone and urogastrone analog products of
the invention are lysed and centrifuged and the super-
natant is applied to the culture of A-431 cells and
incubated. The culture medium is then assayed for
the presence of Il25-labelled urogastrone displaced
from bound association with cell surface receptors
by products of the invention present in the microbial
cell lysate supernatant.
Polypeptide products of the invention which
include amino terminal residues in addition to the
native urogastrone sequence may be processed, if desired,
to remove the additional residues. For example, the
above-noted [Met l]urogastrone may be suitably treated
with cyanogen bromide to yield polypeptides commencing
with an amino terminal asparagine residue characteristic
of the naturally occurring urogastrone products.
If such procedures are to be employed, it may ~e expec-
ted that the [Met2l] residue of urogastrone polypeptide
- 22 -
products might provide an additional site for cyanogen
bromide cleavage or the methionine may be chemically
transformed to a homoserine residue. Alternately,
the methionine residue at position 21 may be replaced
by another amino acid, such as valine, through reconstruc-
tion of the DNA sequence to delete the methionine-
specifying codon, ATG, and replace it with an alternate
codon, such as GT~ which specifies valine. Applied,
e.g., to the construction of Example 4, this process
would involve an initial variation in construction
of oligonucleotide segments 3 and 10. Alternately,
the modification could be efected by excising the
HinfI/~hI fragment from plasmid pADH55 and replacing
it with a manufactured sequence including the desired
codon change. The cyanogen bromide cleavage product
of microbial expression of such an altered gene would
itself be an analog of urogastrone, e.g., [Val21]urogas-
trone.
Products of t`ne present invent~ion and/or
antibodies thereto may be suitably "taggèd", for example
radiolabelled (e.g., with I1253 conjugated with en2ymes
or fluorescently labelled, to provide reagent materials
useful in assays and/or diagnostic test kits, for
the qualitative and/or quantitative determination
of the presence of such products and/or said antibodies
in fluid samples. Such antibodies may be obtained
from the innoculation of one or more animal species
(e.g., mice rabbit, goat, human, etc.) or from mono
clonal antibody sources. Any of such reagent materials
may be used alone or in combination with a suitable
substrate, e.g., coated on a glass or plastic particle
or bead.
Numerous modifications and variations in
the practice of the invention are expected to occur
to those skilled in the art upon consideration of
the fore~oing illustrative examples~ Consequently,
2~473g
- 23 -
the invention should be considered as limited only
to the extent reflected by the appended claims.
