CA1214739A - Manufacture and expression of genes for urogastrone and polypeptide analogs thereof - Google Patents

Abstract

ABSTRACT

"THE MANUFACTURE AND EXPRESSION OF GENES FOR
UROGASTRONE AND POLYPEPTIDE ANALOGS THEREOF"

Disclosed are DNA sequences comprising struc-tural genes coding for (1) a polypeptide having the amino acid sequence and properties of urogastrone and for (2) polypeptide analogs thereof which differ in terms of the identity and/or location of one or more amino acids, e.g., [Asp25] and [Pro52, Pro53]
analogs of urogastrone. Structural gene sequences may be provided with initial and terminal sequences which facilitate production of discrete protein products by selected host microorganisms as well as for expression by host organisms of fusion proteins, e.g., .beta.-lactamase-urogastrone and .beta.-galactosidase-urogastrone from which the desired products may be isolated.

Description

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~

[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 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 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 "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 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;

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.

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.

Oligonucleotide fragments were synthesized using a four-step procedure and several intermediate washes. Polymer bound dimethoxytrityl protected nucleo-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 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.

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

(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

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-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-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-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 ~.

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 -- 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~

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.

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~

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~

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~ ~:

- 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~

The following example relates to presently preferre~ procedures for enhancing the levels of expres-sion of products of the invention.

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~

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 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 the invention should be considered as limited only to the extent reflected by the appended claims.

Claims (22)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A manufactured gene capable of directing the synthesis in a selected host microorganism of a urogastrone polypeptide analog which differs therefrom in terms of the identity and/or location of or the addition of one or more amino acids.

2. A manufactured gene according to claim 1 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.

3. A manufactured gene according to claim 2 wherein the base sequence includes one or more codons, selected from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in E. coli.

4. A manufactured gene according to claim 1 wherein the base sequence comprises one of the following:

5'-ATG AAC TCT GAT TCC GAA TGC CCG CTG TCC-3'-TAC TTG AGA CTA AGG CTT ACG GGC GAC AGG-CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CGA CAG CTG TTT ATG CGA ACG TTG ACG

GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-GAT CTG AAA TGG TGG GAA CTG CGT-3' CTA GAC TTT ACC ACC CTT GAC GCA-5' and 5'-ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT
3'-TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA

TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT
ATG ACG CAG GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA

CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC
GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG

GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA
CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT

CTG CGT-3' GAC GCA-5' and 5'-ATG AAG AAA TAT TGG ATC CAA
3'-TAC TTC TTT ATA ACC TAG GTT

ATG AAC TCT GAT TCC GAA TGC CCG CTG TCT CAT GAC GGT
TAC TTG AGA CTA AGG CTT ACG GGC GAC AGA GTA CTG CCA

TAC TGC CTG CAT GAT GGC GTA TGC ATG TAC ATC GAA GCT
ATG ACG GAC GTA CTA CCG CAT ACG TAC ATG TAG CTT CGA

CTG GAC AAA TAC GCA TGC AAC TGT GTT GTA GGT TAC ATC
GAC CTG TTT ATG CGT ACG TTG ACA CAA CAT CCA ATG TAG

GGC GAA CGT TGC CAG TAT CGC GAC CTG AAA TGG TGG GAA
CCG CTT GCA ACG GTC ATA GCG CTG GAC TTT ACC ACC CTT

CTG CGT-3' GAC GCA-5' and 5'-AAC TCT GAT TCC GAA TGC CCG CTG TCC-3' TTG AGA CTA AGG CTT ACG GGC GAC AGG-CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-TAT ATC GAA GAT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CTA GAC CTG TTT ATG CGA ACG TTG ACG-GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-GAT CTG AAA TGG TGG GAA CTG CGT-3' CTA GAC TTT ACC ACC CTT GAC GCA-5' and 5'-AAC TCT GAT TCC GAA TGC CCG CTG TCC-3'-TTG AGA CTA AGG CTT ACG GGC GAC AGG-CAC GAC GGT TAT TGT CTG CAC GAC GGT GTT TGC ATG-GTG CTG CCA ATA ACA GAC GTG CTG CCA CAA ACG TAC-TAT ATC GAA GCT CTG GAC AAA TAC GCT TGC AAC TGC-ATA TAG CTT CGA GAC CTG TTT ATG CGA ACG TTG ACG-GTA GTT GGT TAC ATC GGT GAA CGT TGC CAG TAC CGT-CAT CAA CCA ATG TAG CCA CTT GCA ACG GTC ATG GCA-GAT CTG AAA TGG TGG GAA CCG CCA-3' CTA GAC TTT ACC ACC CTT GGC GGT-5'.

5. A manufactured gene according to claim 1 wherein the base codons specifying urogastrone are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides a recognition site for restriction endonuclease cleavage of a DNA sequence.

6. A fusion gene comprising a manufactured gene according to claim 1 fused to a second gene capable of directing synthesis of a second polypeptide in a manner permitting the synthesis of a fused polypeptide including urogastrone polypeptide or a urogastrone polypeptide analog.

7. A fusion gene according to claim 6 wherein said second gene is a gene directing synthesis of .beta.-galactosidase enzyme.

8. A fusion gene according to claim 6 wherein said second gene is a gene directing synthesis of .beta.-lactamase.

9. A biologically functional DNA microorganism transformation vector including a manufactured gene according to claim 1.

10. A biologically functional DNA microorganism transformation vector including a fusion gene according to claim 6.

11. A vector according to claim 9 or 10 which is a circular DNA plasmid.

12. A microorganism transformed with a vector according to claim 9 or 10.

13. A process for the production of urogastrone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA
including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce urogastrone polypeptide.

14. A process according to claim 13 wherein -the microorganisms grown are E. coli microorganisms.

15. A urogastrone polypeptide analog produced by the process of claim 13.

16. A process for the production of urogastrone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA
including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce [Asp25] urogastrone.

17. A process for the production of urogastrone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA
including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce [Pro52, Pro53]
urogastrone.

18. A process for the production of urogastrone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA
including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce [Met-1] urogastrone.

19. A process for the production of urogastrone polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA
including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce [Met-8 , Lys-7, Lys-6, Tyr-5 , Trp-4 , Ile-3 , Gln-2 , Met-1] urogastrone.

20. A process of producing a reagent material comprising introducing a radiolabel into the polypeptide analog of claim 15.

21. A process according to claim 20 wherein the radiolabel is I125.

22. A process of producing a reagent material comprising raising an antibody to a polypeptide analog of claim 15, and coating said antibody on a plastic bead.