AU653458B2 - Ovine growth hormone gene and an expression thereof in E. coli - Google Patents

Description

'~?~rglla 653458

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant(s): Actual Inventor(s): LUCKY LIMITED Joong Myung Cho Young Woo Park Kook Jin Lim r *4 a Address for Service: Invention Title: CULLEN CO., Patent Trade Mark Attorneys, 240 Queen Street, Brisbane, Qld. 4000, Australia.

OVINE GROWTH HORMONE4AND AN EXPRESSION THEREOF IN E. COLI 4* The following statement is a full description of this invention, including the best method of performing it known to us: la- OVINE GROWTH HORMONE GENE'AND AN EXPRESSION THEREOF IN E. COLI Field of the Invention The present invention relates to an ovine growth hormone gene and an expression thereof and purification of an expressed ovine growth hormone; and, more specifically, to an ovine growth hormone gene useful for the mass production of ovine growth hormone in E. coli, an expression vector thereof, E. coli cells transformed with said expression vector, a process for producing ovine growth hormone by employing the E.

coli transformants and a process for purifying the ovine growth hormone produced in E. coli cells.

Backaround of the Invention 0 *0 0 C Hitherto, in the stock breeding, the efficiency of the feed and the weight increase of the stocks have been 20 -heightened by way of mixing a conventional feed with a high protein feed or a steroid hormone. However, sources for the high protein feed are limited; and the use of the steroid hormone has become restricted since long residence time of the steroid material in the stocks may adversely affect human bodies.

In contrast, animal growth hormones wh ch are recently 5 20 developed do not remain in the bodies; and are speciesspecific and thereby are not regarded as having any influence on human bodies. Accordingly, animal growth hormones are recognized as an ideal material for the enhanced growth of animals.

Particularly, it has been known that if the growth hormone is administered to ovine, the weight of the ovine and the efficiency of the feed are increased; and the growth of the wool is facilitated.

Animal growth hormone, which is one of the proteins that regulate the metabolisms of lipid and carbohydrate as well as protein and facilitate the growth of tissues, is a single strand globular protein and comprises about 190 to 200 of amino acids [see: Barrington et al., Academic Press, N.Y., 2(1979)].

The growth hormone which is produced in an anterior pituitary is matured from a precursor which has a signal sequence at the amino terminal by cleavage of the signal sequence when passing through an endoplasmic reticulum(Davies et al., Nature 283, 433-438(1980)).

The growth hormone facilitates a balanced growth of animals by way of promoting the transmission of amino acids into the cells and stimulates the translation of mRNA; and promotes the fission of the cells which is an important factor for the growth. Therefore, the weight of the animal injected with the growth hormone is additionally increased without an .1 ricroii)inL of tho kooJd a it r:u1 of i t1.1m1o (I "to protein.

The entire sequences of most animal growth hormones have already been known. Also, some animal growth hormone genes including human, bovine, pig and rat have been cloned and expressed in E. coli(Miller et al., JBC 255, 7521-7524; Martial et al., Science 205, 602-607(1979); Seeburg et al., Nature 270, 486-494(1977)). However, hitherto ovine growth hormone has not yet been mass-produced in E. coli by employing a genetic engineering method.

Summary of the Invention Accordingly, it is an object of the present invention to 15 provide an OGH gene which is designed to be efficient and :useful for the ;lass-production of OGH in E. coli.

It is another object of the present invention to provide an expression vector comprising said OGH gene and E. coli cells transformed with the expression vector.

20 It is a further object of the present invention to provide a process for producing OGH by employing the E. coli transformants.

It is still another object of the present invention to provide a process for isolating and purifying OGH produced in 25 the E. coli transformants efficiently.

Accordingly, in accordance with one aspect of the present 4 invontion, thloo i o providod on 001G ono having a nuclooti do iscquonco laown in Fi'.g. I which i dooignod to p)Oduco maturo OUGI in E. coli.

In accordance with another aspect of the present invention, there is provided an expression vector comprising said OGH gene, and E. coli cells transformed with said vector.

In accordance with a further aspect of the present invention, there is provided a process for producing OGH in E.

coli comprising culturing the E. coli transformants under a culture condition appropriate for the expression of the OGH gene.

In accordance with still another aspect of the present invention, there is provided a process for purifying OGH which comprises: isolating an insoluble material from an E. coli transformant in which a Anature OGH is produced and dissolving the insoluble material in a buffer capable of dissolving the material; centrifuging the dissolved material to obtain a precipitate; lissolving the precipitate in a buffer capable of dissolving same and passing the resultant solution through an anion exchange resin with a NaCl solution to obtain an eluate; :.2Q concentrating the eluate; and subjecting the concentrate to gel permeation chromatography.

Brief Description of the Drawings The invention can be more readily understood by reference a a e

OS.

to tho aC c nomp ny ing (dlrdaw.lnyu,, whlioi'tln Fig. 1 shows the nuclootide sequence of an OGH gene of the present invention and the amino acid sequence derived therefrom; Fig. 2 describes the scheme for obtaining an OGH gene; Fig. 3 illustrates the scheme for constructing an expression vector comprising the OGH gene for the expression of the OGH gene in E. coli; Fig. 4 offers the result of SDS-PAGE(sodium dodecylsulfate-polyacrylamide gel electrophoresis) of the cell extract of E. coli transformants cultured under the condition appropriate for the expression of the OGH gene; Fig. 5 represents the scheme of isolation and purification for OGH from the E. coli transformants; 15 Fig. 6 discloses the result of the electrophoresis of the S* insoluble protein sample from the E. coli transformants; Fig. 7 depicts the result of DEAE anion exchange chromatography of the protein sample after dissolving the insoluble proteins; 20 Fig. 8 presents the result of S-200 gel permeation chromatography of the protein sample after DEAE anion exchange chromatography; and Fig. 9 demonstrates the bioactivity of the purified recombinant OGH.

DoLallaid DoulrIPLion, ur Wlin .l.uVvoI!,-lD.

The OGH gene of the present invention is designed on the basis of the amino acid sequence of OGH as described by H. Li et al.(Biochem. Biophys. Acta. 29, 493(1973)), as follows: Phenylalanine, an N-terminal amino acid of mature OGH, is introduced into the next position to an initiation 'codon, a methionine, and a recognition site of a restriction endonuclease NdeI(5'-CATATG-3') is generated by introducing 5'-CAT-3' at the front of the initiation codon(ATG); (ii) Two termination codons TAG and TAA are introduced into the next position to a phenylalanine which is a C-terminal amino acid of mature OGH so that the translation is terminated 15 at said phenylalanine, and a recognition site of a restriction endonuclease SalI(5'-GTCGAC-3') is introduced to OGH gene so as to facilitate a cloning of the gene into an expression Svector; (iii) The recognition sites of several restriction 20 endonucleases, SacI(5'-GAGCTC-3'; arginine which is the

S.

17th amino acid), PstI(5'-CTGCAG-3'; leucine which is the amino acid) and XbaI(5'-TCTAGA-3'; leucine which is the 116th :amino acid), are introduced into the gene so as to facilitate a.modification of the OGH gene; and 25 (iv) The nucleotide sequence or subsequences which are capable of forming a secondary structure are modified by substitution

'I

of( ooimlain nuGoloot1(do t nt u m lu IiuI I nii H p N IN 1'i.,i at the QIOVO o' transcripltioI and tlan~ latlon.

The entire nucleotide sequence of an OGH gene my be synthesized in accordance with a conventional method knowr in the art, a method using DNA synthesizer or polymerase cha'n reaction(PCR).

An expression vector of the present invention to express said OGH gene in E. coli may be prepared by employing various expression systems operable in E. coli.

For instance, such a vector may be prepared by using a vector ptrp322H-HGH described in Korean Laid-open Patent Publication No.91-457(E. coli W3110 containing said Dtrp322H- HGH has been deposited with Korean Collection for Type Cultures, with the accession number of KFCC-10667).

15 Specifically, the vector is prepared by substituting an OGH gene shown in Fig. 1 for human growth hormone gene in ptrp322H-HGH, as illustrated in the Examples given below.

An expression vector containing the OGH gene so obtained is named as ptrp-OST and E. coli W3110(ATCC 27325) transformed 20 therewith was deposited with Korean Culture Center of Microorganisms on June 26, 1991, with the accession number of KCCM-10006 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.

25 An E. coli cell transformed with an expr"ssion vector containing an OGH gene is then cultured under the condition Crtl)iilo ol (lyiprti.tKi l l (II t (jo whifh ti l r l. n l according to tho chractor'lstics of tho ve ct, and the host E. coli cell.

For example, E. coli W3110 cell transformed with. an expression vector ptrp-OST is cultured in M9 medium which contains ampicillin and tryptophan. When the optical density (OD) value at 600nm reaches 0.7, indoleacrylic acid is added thereto, followed by further cultivation.

The OGH produced in the E. coli transformant may be isolated and purified by a combined use of conventional methods, cell disruption, centrifugation, dialysis, salting out, chromatography, gel filtration, electrophoresis and electroelution; however, it is necessary to choose a most appropriate method and condition for obtaining highly purified 15 OGH in mass, as is shown here.

For the cultured E. coli W3110(ptrp-OST), the culture is centrifuged; and the pre pitated cells are suspended in a Tris buffer and treated with an ultrasonicator to disrupt the cells. The disrupted cells are centrifuged and the precipitates are collected.

The precipitates dissolved in the Tris buffer is centrifuged. The supernatant is passed through an anion exchange resin column which is equilibrated with an ammonium carbonate buffer. Th' protein adsorbed in the column is isolated with a concentration gradient of 0-0.5M NaC1.

Accordingly, OGH is eluted at 0.1M to 0.2M NaC1.

9 Each of the eluted fractions is subjected to electrophoresis and. the OGH fraction so collected is concentrated by using an ultrafiltration membrane; or is first precipitated by adding ammonium sulfate to make a saturated concentration, followed by further concentration process. The concentrate is passed through an S-200 resin column and the fractions having a purity of at least 95% are collected.

The following examples are intended to specifically exemplify the present invention without limiting the scope of the invention; and the experimental methods used in Examples are practised in accordance with Reference Examples given hereinbelow unless otherwise stated.

Unless otherwise specified, percentages given below for 15 solids -i solid mixtures, liquids in liquids and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, •respectively.

e 20 S. c..

Reference Example 1: Dicestion of DNA with restriction endonuclease The restriction enzymes and reaction buffers used herein were purchased from NEB(New England Biolabs, Jolla, MA, The reaction was generally carried out in a sterilized Eppendorf tube with a reaction volume ranging from 50 to 10 100~1, at a temperature of 37 0 C for 1 to 2 hours. Thereafter, the reaction mixture was heat-treated at 65 0 C for minutes(or extracted with phenol and precipitated with ethanol in the case of a heat-resistant endonuclease) to inactivate the restriction endonuclease.

x reaction buffer for the reaction of a restriction endonuclease had the following composition: x NEB reaction buffer 1: 100mM bis Tris propane-HC1, 100mM MgCl 2 10mM dithiothreitol(DTT), pH 0 10 x NEB reaction buffer 2: 100mM Tris-HCl, 100mM MgC1 2 500mM NaC1, 10mM DTT, pH x NEB reaction blffer 3: 100mM Tris-HCl, 100mM MgC12, 1000mM NaC1, 10zia DTT, pH 7.0; and x NEB reaction buffer 4: 200mM Tris-acetate, 100mM 5 magnesium acetate, 500mM potassium e 3tate, 10mM DTT, pH 1

S

S

*5 S* Reference Example precipitation 2: Phannl axtraction and ethanol 2 Phenol extraction 20 After the completion of the enzyme reaction, the reaction mixture was extracted with phenol for che purpose of inactivating the enzyme or recovering the DNA in the reaction mixture, wherein phenol preequilibrated with a buffer containing 10mM Tris-HCI(pH 8.0) and ImM EDTA(ethylenediaminetetraacetic acid) was used. Phenol extraction was carried out by mixing equal volumes of the sample and the phenol with 11 vigorous shaking; centrifuging the mixture at 15,000rpm for minutes; and transferring the aqueous layer into a new tube.

The above procedure was repeated two or three times.

The aqueous layer was, then, extracted with an equal volume of chloroform solution(chloroform isoamyl alcohol 24:1) and the aqueous layer was separated again; 0.1 volume of 3M sodium acetate and 2.5 volumes of e-chanol were added thereto; and, the mixture was centrifuged at 15 000rpm and 4 0

C

for 20 minutes after having left it at -70 C for 30 minutes or at -20 0 C for 12 hours, to recover the nucleic acid.

Reference ExamDle 3: Lication reaction 15 ooo oo 6 Ligation reaction of DNA was carried out by employing T4 DNA ligase and 10 x ligation reaction buffer(pH 7.8, Tris-HCl, 0.1M MgC 2 1, 0.2M DTT, 10mM ATP, 0.5mg/ml bovine serum albumin(BSA)) purchased from NEB. The reaction volume was generally 20Ll, and 10 units of T4 ligase was used for the ligation of cohesive ends of DNA while 100 units were used for the ligation of blunt ended DNAs.

The reaction was carried out at 16 0 C for 5 hours or at 4 0 C for over 14 hours; and, after the reaction was completed, the reaction mixture was heated at 65 0 C for 15 minutes to inactivate the T4 DNA ligase.

4* *4*s Sr

S.

S

20 Reference Ex mple 4: Transformation of E. coli -12 E. coli strains used for the following examples included E. coli HBlOl(ATCC 33694), E. coi W3110(ATCC 27325) and E.

coli JM105(ATCC 47016). Transformation of E. -coli was carried out by employing a method known in the art, as described in Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y.(1982), or by Cohen in Proc.

Natl. Acad. Sci. 69, 2110(1972).

Reference Example 5: Synthesis of olicronucleotide 15 *0 C

C

C

S

C

C.

S

C. C

C

C.

o 25

VI

01ligonuc leot ides were synthesized by employing a DNA synthesizer (Applied Biosystems Inc., 380B, of automatic solid phase phosphoamidite cheinistry.

The synthesized oligonucleotides were purified by using denaturing polyacrylamide gel(2M urea, 12% acrylamide and bisacrylamide(29:1), 50mM Tris, 50mM boric acid, 1mM EDTA) electrophoresis and SEP-PAK(Waters Inc., U.S.A) column chromatography; and the amount was determined by measuring the O.D. value at 260nm.

Reference Example 6: Polymerase chain reaction(PCR) To a mixture of 10 to lO0ng of a templ~ate DNA, 10kLi of x Taq polymerase reaction buffer(lOmM Tris-HCi., 500mM KCl, 15mM MgCl 2 gelatin, pH 1041 of a mixture of dNTP's(eaich of dGTP, d.ATP, dTTP and dCTP is 1.25mM), 24~g of 13 each primer(generally, 2 primers were used For the reaction, and in the case that 4 primers were used, the primer located in the middle was used in an amount of 0.02.g), and 0..L of Ampli Taq DNA polymerase(Perkin Elmer Cetus, was added distilled water in an amount to make a total volume of 100pl; and 501i of mineral oil was added thereto to protect the reaction mixture from evaporation.

The PCR was carried out by ubing a thermal cyoler(Perkin Elmer Cetus, and the thermal cycle was programmed to repeat appropriately, the cycle being: 950C for 30 seconds 0 C for 30 seconds 72 0 C for 1 minute; and, finally, *the reaction was carried out at 72 0 C for 10 minutes.

After the reaction was completed, the mixture was extracted with phenol and the PCR products were recovered by precipitation with ethanol; and, the precipitate was dissolved in 20~p of TE buffer solution(10mM Tris-HC1, ImM EDTA, pH oxamz1e Preoaratlon of QGH gane Desg.an and synthesis of the oliqconuoleotideg for econstructiag an OGH ce4e In order to prepare an OCH gene containing a recognition *site of a restriction endonu7)ease NdI at the 5'-end and two o termination rodon o and a recognition site of a restriction :endonuclease Sall, the following 10 oligonucleotides were -14 synthesized by employing the same method as described in Reference Example OGH 1: 5 '-CCCCATATGTTCCCGGCTATGTCTCTATCTGGTCTATTCGCTAACGCTGTT CTTCGAGCTCAGCATCTTCATCAGCTGGCTGCTGA-3' OGH 2: 5 '-TTCTGGATGGAGTATCTCTGTCCCTCCGGGATGTAGGTGCGCTCAAACTC TTTGAAGGTGTCAOGCAGCCAGCTGATGAAG-3' 0GM 3: 5 '-CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCTGAAACC ATCCCGGCCCCCACGGGCAAGAATGAGGC- 3' OGH 4: 5 '-AGCCACGACTGGATCAGGAGCAGTGAGATGCGAAGCAGCTCCAAGTCTGAT TTCTGCTGGGCCTCATTCTTGCCCGTGGG-3' OGH- 5: 5 '-CTCCTGATCCAGTCGTGGCTCGGGCCCCTGCAGTTCCTCAGCAGAGTCTTC ACCGACAGGTTGGTGTTTGGCACCTCGGA-3' 0GM 6: 5 '-AGCTCCCGCATCAGGGCCAGGATGCCTTCCTCTAGATCCTTCAGCTCTCTCA TAGACACGGTCCGAGGTGCCAAACACCAA-31 15 0GH 7: 5 '-CTGGCCCTGATGCGGGAGCTGGAAGATGTCACCCCCCGGGCTGGGCAGATC CTCAAGCAGACCTATGACAAATTTGACAC- 3' 0GM 5 -TTCCGGAAGCAGGAGAGCAGACCGTAGTTCTTGAGCAGCGCGTCGTCACTG CGCATGTTTGTGTCAAATTTGTCATAGGT- 3' 0GH 9: 5 '-CTGCTCTCCTGCTTCCGGAAGGACCTGCATAAGACGGAGACGTACCTGAGG GTCATGAAG3' OGH 10: 5 r-GGGGTCGACTACTAGAAGGCGCAGCTGGCCTCCCCGAAGCGCGGCACTT CATGACCCTCAGGTACG- 3' Oligonucleotide OGHi contains a recognition site of the restriction endonuclease NdeI, 5 -CATATG-3', at the and, 20 nucleotides at the 3 '-end are complementary to those at the 5'-end of OGH-2.

nucltootides at the 5 '-end of OG1H3 are complementary to those at the 3'--end of OGH4; 20 nucleotides at the 5'-end of 00H5 are complementary to those at the 3'-end of 0GH6; nucleotides at the 5'-end of 00H7 are complementary to those at the 3'-end of OGH8; 20 nucleotides at the 5'-end of OGH9 are complementary to those at the 3'-end of OGH 10; and contains two termination condons 5'-TAG-3' and a recognition site of the restriction endonuclease Sail, 5'-GTCGAC-3' to facilitate the cloning into an expression vector.

Preparation of entire OG- grene <Step 1> 15 o 0 0 S. *e U U 20 0* *0 0 0 4* .55.*0 0

S

A first PCP. was carried out as follows, referring to Reference Example 6. 2kLg of OGHI, 0.024~g of OGH2, 0.024~'g of OGH3 and 2jJ~g of OGH4 were added to a reaction tube A; 2 Lg of OGH5, 0.021.kg of OGH6, 0.024g of OGH7 and 24~g of 0GH-8 were added to a reaction tube B; and 2kLg of OGH9 and 2JJtq of 001-11 were added to a reaction tube C. To each of the reaction tubes were added 101-l of 10 x Taq polymerase buffer (100mM Tris-HCl, pH 8.3, 500mM KCl, 15mM MgC1 2 0. gelatin), 10j41 of dNTP's mixture(1.25mM dGTP, dATP, dTTP and dCTP), 0.541l(5 units/4l) of Ampli Taq DNA polymerase and 6741.L of distilled water. The thermal cycle of 95WC for 30 seconds 0 C for 30 seconds 720C for 1 minute was repeated 4 times, 15 0@ 0 90 00 0 00 0 0 0 00 0000 0 000.

00 00 .0 0 P0 20 00 %0 0900 09 0 and tho roaction was -Cur thor clarr tod out o t 7211C fo! minutes. The result-ing mixture was subjected to polyacrylamide gel electrophoresis to isolate a first PCP.

product. The product was purified by using an Eiutip- D(Schleicher Schuell, and dissolved in 2041l of TE buffer(lOmM Tris-Ci, pH 7.5, 1mM EDTA).

<Step 2> A second PCR was carried out by using the first PCR product as follows.

About 1b0ng of the PCR product in the reaction tube A, about 1b0ng of the PCR product in the tube B, 2Jpg of OGI, 2kLg of OG-18, 10pLl of 10 x Taq polymerase buffer, l1gil of dNTP's mixture, 0.5k.Ll(5 units/kil) of Ampli Taq DNA polyrnerase and 67ptl of distilled water were added to a reaction tube D; and the mixture was subjected to PCR 20 times. The second POR product thus obtained was treated in the same manner as in Step 1 and dissolved in 2041.L of TE buffer.

<Step 3> A third PCR was carried out by using the second PCR product as follows.

About lO0ng of the PCR product in the reaction tube D, about lO0ng of the PCR product in the tube C, 2gig of OGHi, 21-Lg of OGH 10, 1041.v of lOx Taq polymerase buffer, 1041 of dNTP's mixture, 0.51JLl(5 units/t.b) of Ampli Taq DNA pobymerase and 39 *0 000t00 0 17 674l of distilled water were added to a reaction tube E; and the mixture was subjected to PCR.20 times. The third PCR product thus obtained was treated in the same manner as in Step 1 to obtain a fragment(hereinafter, referred to as "fragment OGH") containing an entire or full length OGH gene which was dissolved in 20p.l of TE buffer. The entire procedures to prepare a fragment OGH are described in Fig. 2.

Example 2: Preparation of expression vector Preparation of fragments OGH-N/L, PL and PN About 2pg of a fragment OGH obtained in Example 1 was digested with a restriction endonuclease NdeI(10 units) at 37 0 C in 50Ll of 10 x NEB reaction buffer 4 and subjected to 15 phenol extraction and ethanol precipitation. The precipitated nucleic acids were dissolved in 51J.l of 10 x NEB reaction buffer 3 and treated with the restriction endonuclease units) for 2 hours at 37 0

C.

On the other hand, 2kg of plasmid ptrp322H-HGH(see Fig.

20 3) which was isolated from E. coli W3110(KFCC-10667) was "digested with the restriction endonucleases PstI and SalI in the same manner as in the above; another 2yg of plasmid ptrp322H-HGH was digested with the restriction endonucleases PstI and NdeI. The digested plasmids were subjected to 0.7% agarose gel electrophoresis to isolate 0.6Kb, 1.5Kb and 0.8Kb of fragments, which were named fragments OGH-N/L, PL and PN, 18 respectively.

Lication of fragments OGH-N/L, PL and PN To a mixture of 100ng of fragment OGH-N/L, 100ng of fragment PL and 100ng of fragment PN were added 2J.l of ligase buffer and 10 units of T4 DNA ligase; and distilled water was added thereto to adjust the total volume to be 2C-01.

The resulting mixture was reacted at a temperature of 1i6°C for 12 hours. After the completion of the reaction, the resultant was transformed into E. coli HB101(ATCC 33694) and ptrp-OST containing a fragment of OGH gene '-as selected. The ptrp-OST was subjected to dideoxy DNA sequencing(Sanger, F. eL P.N.A.S. 74, 5463(1977)) by employing a primer 2090(5'- (CATCACCGAAACGCGCGAG-3') and a primer ptrpl(5 '-GACAATTAATCATC 15 GAACTA-3') to confirm the nucleotide sequence of the entire OCH gene.

Example 3: Transformation of E. coli with expression vector and expression of OGH aene o ae oo E. coli W3110(ATCC 27325) was transformed with the plasmid ptrp-OST obtained in Example 2 in accordance with the method of Reference Example 4; and the transformant was cultured with shaking in LB medium(10g of Bacto-triptone, 5g of yeast extract, 5g of sodium chloride per 1 liter) containing 504g/ml of ampicillin for 12 hours at a temperature of 37 0 C. 5ml of 19 the culture was transferred 'to each of 11 flasks charged with 1L of M9 medium(40mM K 2

HPO

4 22mM KH 2

PO

4 8.5mM NaCI, 18.7mM NHCl, 1% glucose, O.1mM MgSO, 0.1mM CaC12, 0.4% casamino acid, 10mg/ml of Vit. B 1 containing 40.g/ml of ampicillin and cultured with shaking for about 3 to 4 hours at 37 0

C.

Indoleacrylic acid(IAA) in various concentrations(from OmM to with an increment of 0.1mM) was added to express an OGH gene when the O.D. value of each culture reached 0.5 at 650nm.

After about 5 hours from the addition of IAA(and after 12 In to 24 hours from the cultivation in case that IAA was not added), the cell culture was centrifuged at 3,000rpm tor minutes with a centrifuge(Beckman J6-B, Rotor JS 4.2) to obtain an E. coli cell precipitate. The cell precipitate was suspended in a sample buffer and subjected to electrophoresis 15 on 12% polyacrylamide gel according to Laemmli method(Laemmli, Nature 227, 680(1970)) to identify the expression of the gene.

The result is shown in Fig. 4.

Lane 1 of Fig. 4 shows a protein gel pattern of the E.

coli cell sample which does not contain an OGH gene; lanes 2 20 to 9 show protein gel patterns of the E. coli cell sample which contains an OGH gene when IAA was added in the concentration of 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM and 0.8mM, respectively.

As can be seen from Fig. 4, it was found tnat OGH was expressed in view of the clear bands exhibited at about 21Kd position in lanes 2 to 9.

20 Example 4: Purification of OGH expressed in E. coli Culture of E. coli transformant Among the E. coli cultures of Example 3, the E. coli culture that induced the expression of OGH gene at 0.2mM IAA was employed for further purification. The cell culture was centrifuged at 3500rpm for 30 minutes with a centrifuge (Beckman J-6B, Rotor JS4.2) to obtain an E. coli cell precipitate.

e e j 15 e 15 *o r a '.:20 Disruption of cell and isolation of insoluble protein 4g of the E. coli cell precipitate obtained in (4-A) above was suspended in 30ml of a buffer(50mM Tris, pH 8.0, EDTA, 5mM 13-mercaptoethanol) at The suspension was subjected to ultrasonication in an ice-bath for 20 minutes with an ultrasonicator(Heat-systems-Ultrasonics Inc., W225, to disrupt the cell to be a homogenate.

To the homogenate of E. coli cells thus obtained was added 150ml of said buffer, followed by stirring for 1 min.

Thereafter, the solution was centrifuged at 5,500rpm for minutes with a centrifuge(Beckman J2-21, Rotor JA-14). The supernatant was discarded; and the precipitate was suspended in 200ml of a buffer(50mM Tris, 1M NaC1, 5mM EDTA, 5mM 3mercaptoethanol). The suspension was stirred for 10 minutes and centrifuged again at 5,500rpm with the centrifuge. The supernatant was discarded and the precipitate was suspended in 21 200m. of a buffer(5OmM Tris, 4M urea, 2mM BDTA, 5mM /3nercaptoethanol). -The suspension was stirred for 10 minutes at a room temperature and centrifgei at 8,000rpm for minutes with'a centrifuqe(Beckman J2-21, Rotor JA14) to obtain ~an insoluble protein precipitate.

IlPnosJluj of insoluble OGE To the insoluble protein precipitate obtained In (4-B) above war added 20m1 of bufer(l0 m Tris, -i 8.0, 8M uroa, EDTA, 5mM DTT), followed by-stirring for 2 hours at a room temperature. The resulting solution was centrifuged at 15,000rpm for 20'minutes with a centrifuge(Bekman J2-21, Rotor JA2l) to remove the insoluble materials. The supernatant in which OGH was dissolved was recovered and amtionium carbonate butfFer(PH 9.0) was added thereto in an amount to make a urea concentration of 'Mf followed by stirring for I hour. The solution was further xentrifgec at 15,000rpm for 20' minutes to remove the precipitate. T8 supernatant was subjected to electrophoresi; 4nd the result is shown in FIJg. 6, Lane 1 of Fig c shows the cell homogenate; lane-, 2 shows the disrupted cell precipitate; lane 3 shows the precipitrate washed with the bvvfer containing IM NaCl; lane 4 shows the supernatant obtained by suspending the precipitate with the buffer containing 4M urea and centrl qgAng the same; lane shows the precipitate washed with the buffer containiFng' 4M urea(The samples of Janes 1 to 5 were obtained in Example (4- 22 lane 6 shows the precipitate which was not dissolved in the buffer containing 8M urea; and lane .7 shows the protein dissolved in the buffer containing 8M urea.

Anion excbanae resin colgumn chrgmatographli The supernatant obtained in abovo was panced at a flow rate of 10ml/cm/hr through a DE52 ion exchange resin column (2.5cm x 10cm, Whatman, equilibrated with a buffer.(pH 9.6, iM urea, 5mM ammonium carbonate). The materials remaining 'in the column in free form and urea were thoroughly washed with 5mM ammonium caroonate(pH The proteins adsorbed in the column were eluted with a linear concentration gradient of OM to 0.5M NaC1 in 5mM ammonium carbonate. OGH was eluted at O.IM to 0.2M concentration6 of S. NaC1.

S 15 5-200 ael permeat1;n chromatography 120ml of the OGH solution obtained in above was passed through YM10 Ltrafiltration membrano(Amicon, U.S.A.) to concentrate so as to make a volume of 20ml. Alternatively, to the OGH solution was added ammonium sulfate to make a saturated solution, followed by centrifuging and dissolving the precipitate in 20m1l of a buffer(pH 9.6, 0.25mM NaC1, ammonia carbbnate). The concentrate was passed at a flow rate of 10ml/cm 2 /hr through an S-200 resin column(Pharmacia, .S weden, 5cm x 100cm) equilibrated with a bufferlpH 9.6, 0.25M NaC1, 10mM ammonia carbonate) to isolate the protein.

Oetermination of bioactivitv of OGH 23 To determine the bloactivity of the OGH4 obtained in (4-2) above, the 0GW was injected subcutaneously to mice subjected to a pituitectomy and their weigbit Increase was measlured as follows, 4 to 5 days old femnale mice(species: WJiSter) which were subjected to a pituitectomy were obtained from Charles River Lab located in and the 0GH solution obtained in (4-E) above was passed through a pyrogen adsorption filter(Cuno, U.S.A.)-to remove the pyrogen before use.

Five of such mice -were injected subcutaneously with of physiological saline containing 0.2mg/mi of OGHfo'( 14 days, respectively; and a second batch of five mice were injected with 0.5mlfciay of physiological saline without 0GH for 14 days, 15 On the 14th day, those Injected with O showed an averagje weight increase of 2,3g which corresponded to~ a inqrease over the original weight, As can be seen from the result, OGLI expressed in accordance with the present invention exhibits a high bloactivity.

While the invention has been described with respect to Sthe abovb specific embodiments, it should be recoqnized that various modifications and changes which may be apparent to those skilled in %'he art to which the invebntion pertains may be made and also fall within the scope of the invention as defined by the claims that follow.

Claims (10)

1. An ovine growth hormone gene having the following nucleotide sequence: ATG TTC CCG GCT ATG TCT -CTA TCT GGT Met Phe Pro Ala Met 'Ser Leu Ser Gly T Leu CAG Gin CCC Pro CTC Leu TTG Leu CTG Leu ACC Th~r CTG Le u CTG Leu TCC qer AAG Lys CAG Gin CGC Gly ATG Met AAA Lys TC Cys GAC Asp AAC Asn GCC 'Ala CTC Leu GAO Asp CTG Leu ACA Thr AAG Lys GCC Ala ACC Thr ACC Thr Gin GGG Gly CGT Arg GIA GlU AAC Asn GAC Asp AGC s er CTA Leu 90 AAA Lys 150 GTT Val 210 AAA Lys 270 CTG Leu 330 TAT Tyr 390 GTC Val 450 CGC Ai-g 510 CAT His 570 GCC Ala T'rC GCT AAC GCT GTT CTT CGA GCT CAG Phe Ala Asn Ala Val Leu Arg Ala Gin GAG TTT GAG CGC ACC TAC ATC CCG GAG Glu Phe Glu Arg Thr Tyr Ile Pro Giu GCC TTC TGC TTC TCT GAA ACC ATC CCC Ala Phe Cys Phe Ser Giu Thr Ile Pro TCA GAC TTG GAG CTG CTT CGC ATC TCA Se~r Asp Leu Glu Leu Leu Arg Ile Ser CAG TTC CTC AGC A GA GTC TTC ACC GAC Gin Phe Leu Ser Arg Val Phe Thr Asp GAG AAC CTC AAC CAT CTA GAG GAA GGC Giux Lys Leu Lys Asp Leu Glu Clii Cly ACC CCC CGG GCT CGG CAC ATC CTC AAG Thr Pro Arg Ala Cly Gin Ile Leu Lys CAT His 120 GGA Gly CC Al a 240 (-TG Leu 300 AGC Ser 360 ATC Ile 420 CAC Gin 480 CGT Gly 540 AAC Lys CTG Leu TAC Tyr TGC CGC CCC TTC Cys Arg Arg Phe

2. An expression vector comprising the ovine growth hormone gene of claim 1, wherein the ovine growth hormone gene is positioned at a site capable of being expressed in an E. coli cell.

3. The expression vector of claim which is ptrp-OST.

4. An E. coli cell transf-rmed with the expression vector of claim 2 or 3. The E. coli cell of claim 4 which is E. coli W3110 transformed with ptrp-OST (KCCM-i3006).

6. A process for producing an ovine growth hormone which comprises culturing the E. coli cell of claim 4 or

7. A process for purifying an ovine growth hormone which comprises: isolating an insoluble material from an E. coli e* transformant in which a mature ovine growth hormone is produced and dissolving the insoluble material in a buffer capable of dissolving the material; centrifuging the dissolved material to obtain a precipitate; z dissolving the precipitate in a buffer capable of dissolving same and passing the resultant solution through an anion exchange resin column to obtain an eluate; r 26 concentrating the eluate; and subjecting said concentrate to gel permeation chromato- graphy.

8. The process of claim 7 wherein the anion exchange resin is DE 52 recin.

9. The process of claim 7 wherein the gel permeation chromatography is carried out by using S-200 gel resin. The process of claim 7 wherein said elution is carried with a NaCl solution. 3 :'15 ti •o• o

11. The process of claim 7 wherein said concentration is carried out with an ultrafiltration membrane.

12. The invention which is substantially described in the specification with reference to Examples. r e r DATED this 18th day of LUCKY LIMITED By their Patent Attorneys CULLEN CO. December 1992 27 Abstract The present invention relates to an ovine growth hormone gene and an expression thereof and purification of an expressed ovine growth hormone; and, more specifically, to an ovine growth hormone gene useful for the mass production of ovine growth hormone in E. coli, an expression vector thereof, E. coli cells transformed with said express.-)n vector, a process for producing ovine growth hormone by employing the E. coli transformants and a process for purifying the ovine growth hormone produced in E. coli cells. *20