WO1991005863A1 - Facteurs de croissance recombinants - Google Patents

Facteurs de croissance recombinants Download PDF

Info

Publication number
WO1991005863A1
WO1991005863A1 PCT/AU1990/000477 AU9000477W WO9105863A1 WO 1991005863 A1 WO1991005863 A1 WO 1991005863A1 AU 9000477 W AU9000477 W AU 9000477W WO 9105863 A1 WO9105863 A1 WO 9105863A1
Authority
WO
WIPO (PCT)
Prior art keywords
growth factor
egf
plasmid
sequence
protein
Prior art date
Application number
PCT/AU1990/000477
Other languages
English (en)
Inventor
Robert John Moore
Original Assignee
Pitman-Moore Australia Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pitman-Moore Australia Limited filed Critical Pitman-Moore Australia Limited
Priority to AU65006/90A priority Critical patent/AU648272B2/en
Publication of WO1991005863A1 publication Critical patent/WO1991005863A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF] (urogastrone)

Definitions

  • EGF epidermal growth factor
  • TGFo ⁇ transforming growth factor
  • EGF has also been found by Moore and colleagues (1982a,b; 1983) to show considerable promise as an agent for defleecing of sheep.
  • recombinant or plasmid-derived mEGF by this method is far too high to permit the development of a marketable product for the routine de-fleecing of sheep.
  • Many other workers have developed methods for production of recombinant epidermal growth factors, (e.g. Oka et al, 1985; Smith et al, 1982; Urdea et al, 1983; Sumi et al, 1985).
  • these are also expensive, and are generally more suitable for products which can command substantially higher prices per unit of EGF, than can EGF for defleecing sheep.
  • Epidermal growth factor is conveyed to young mammals from the mother in the milk and has a role in promoting the physiological maturation of the gut. Administration of epidermal growth factors to young animals may accelerate this maturation and therefore permit rapid early growth and early weaning in piglets and other animals.
  • human epidermal growth factor promotes healing of peptic ulcers, and may also be useful in healing wounds of the skin and eyes.
  • similar applications are contemplated; for example accelerated healing of the wound caused by mulesing of lambs, and healing of ulcers and injuries particularly in horses or dogs.
  • Growth factors are also useful in synthetic or semi-synthetic cell and tissue culture medium formulations, particularly those containing little or no serum.
  • the present invention provides molecules related to, but structurally different from, epidermal growth factors, which have essentially similar biological activities but which can be produced readily at low cost and on a large scale. These features make such molecules particularly appropriate for certain animal health applications, where widespread use but low unit cost are to be anticipated. Furthermore, the invention provides a means of overcoming the problems of previously known methods of synthesis, while continuing to achieve high levels of production in recombinant host cells. Although the invention is described with particular reference to EGF, the strong sequence homology between EGF and TGF indicates that the invention will also be applicable to TGF, which is to be understood to be within its scope.
  • EGF can be produced as a tandemly repeated molecule, and that this product is biologically active without any necessity to cleave fusion proteins or to release native EGF. Moreover, the product can be produced as inclusion bodies in E.coli. _
  • a recombinant DNA molecule whose sequence encodes a protein having the biological activity of an animal 5 growth factor selected from epidermal growth factor (EGF) and transforming growth factor (TGF ) (growth factor sequence) , said recombinant DNA comprising a leader sequence and at least one sequence encoding the growth factor.
  • EGF epidermal growth factor
  • TGF transforming growth factor
  • the TGFoCs are structurally and functionally
  • the growth factor is preferably of mammalian origin, more
  • 20 preferably from sheep, mouse, human or pig.
  • the leader sequence may be derived from a variety of
  • the leader sequence is preferably from 1 to 50 amino acids long, more preferably 12 to 50, even more preferably 15 to 30, and most preferably 21. For the purposes of this specification, 'growth
  • a plasmid comprising a recombinant DNA molecule as described above.
  • step (d) recovering the recombinant plasmid.
  • the DNA sequence in step (a) is a plasmid, more pref rably pEGF3.
  • step (h) digesting the product of step (d) with the same restriction endonuclease
  • the method of synthesis of the plasmid comprises the steps of:
  • restriction endonuclease in steps (g) and (h) is BstEII.
  • Figure 2 illustrates the structure of plasmid pWRL500, which was the starting material for pWRL525;
  • the solution was further diluted by the addition of 20mM ethanola ine pH 9.0 buffer (same volume as that of buffer C). This was followed by ultrafiltration through a 100,000 nominal molecular weight cut-off (NMWC) filter and then by concentration on a 10,000 NMWC filter.
  • NMWC nominal molecular weight cut-off
  • EGF induces a number of physiological responses in young mice. The most striking are an acceleration of tooth eruption and eye-opening; more variable but usually recognizable are changes in skin and hair. Scurfiness of skin, and curling of hair and reduced hair growth are characteristic. All of these effects have been noted with protein pCW9, and the early eye-opening and tooth eruption are consistently similar to those elicited by rec-mEGF. The results are presented in Tables 6 and 7.
  • Protein pCW9 has been administered to sheep in the same manner as rec-mEGF at a range of dose rates. At doses equivalent to those used for rec-mEGF, the same overall effects are seen - a transitory reduction in feed intake, and weakening of the wool fibre which is maximal within a week and permits easy removal of the fleece by hand.
  • the numeral before the hyphen (1-8) refers to the preparation of protein pCW9 used (see text) .
  • the protein pCW9 preparations in treatments 1 to 8 have all been prepared using different purification protocols. All show good activity except preparation 7. It was noted that a heavy precipitate formed in preparation 7 during formulation.
  • the recombinant EGF of this invention is fully comparable in activity to rec-mEGF of the prior art. This, in turn, is comparable to mEGF of natural origin.

Abstract

L'invention se rapporte à un ADN recombinant, qui code pour une protéine ayant l'activité biologique d'un facteur de croissance épidermique ou d'un facteur de croissance de transformation et qui comprend une séquence de tête et au moins une séquence codant pour le facteur de croissance.
PCT/AU1990/000477 1989-10-11 1990-10-04 Facteurs de croissance recombinants WO1991005863A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU65006/90A AU648272B2 (en) 1989-10-11 1990-10-04 Recombinant growth factors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPJ6812 1989-10-11
AUPJ681289 1989-10-11

Publications (1)

Publication Number Publication Date
WO1991005863A1 true WO1991005863A1 (fr) 1991-05-02

Family

ID=3774272

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1990/000477 WO1991005863A1 (fr) 1989-10-11 1990-10-04 Facteurs de croissance recombinants

Country Status (3)

Country Link
CN (1) CN1051198A (fr)
WO (1) WO1991005863A1 (fr)
ZA (1) ZA908042B (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102020710B (zh) * 2010-09-03 2012-09-05 深圳市华生元基因工程发展有限公司 人表皮生长因子一个新的突变体en-46

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0123289A2 (fr) * 1983-04-26 1984-10-31 Chiron Corporation Facteur-A et ses signaux de traitement
WO1986002271A1 (fr) * 1984-10-19 1986-04-24 Chiron Corporation Stimulation de la cicatrisation de plaies a l'aide du facteur de croissance de l'epiderme humain prepare a partir d'adn recombinant
EP0326046A2 (fr) * 1988-01-25 1989-08-02 Takeda Chemical Industries, Ltd. Préparation du facteur de croissance épidermique humain
JPH0213381A (ja) * 1988-06-30 1990-01-17 Wakunaga Pharmaceut Co Ltd 複数発現ベクターおよびこれを用いたタンパク質の製造法
AU3814589A (en) * 1988-07-15 1990-01-18 Nippon Shinyaku Co. Ltd. Process for producing hegf
AU4005289A (en) * 1988-08-25 1990-03-01 Smithkline Beecham Corporation Recombinant saccharomyces
EP0357391A2 (fr) * 1988-08-31 1990-03-07 Allelix Biopharmaceuticals Inc. Excrétion de protéines hétérologues de E. Coli

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0123289A2 (fr) * 1983-04-26 1984-10-31 Chiron Corporation Facteur-A et ses signaux de traitement
WO1986002271A1 (fr) * 1984-10-19 1986-04-24 Chiron Corporation Stimulation de la cicatrisation de plaies a l'aide du facteur de croissance de l'epiderme humain prepare a partir d'adn recombinant
EP0326046A2 (fr) * 1988-01-25 1989-08-02 Takeda Chemical Industries, Ltd. Préparation du facteur de croissance épidermique humain
JPH0213381A (ja) * 1988-06-30 1990-01-17 Wakunaga Pharmaceut Co Ltd 複数発現ベクターおよびこれを用いたタンパク質の製造法
AU3814589A (en) * 1988-07-15 1990-01-18 Nippon Shinyaku Co. Ltd. Process for producing hegf
AU4005289A (en) * 1988-08-25 1990-03-01 Smithkline Beecham Corporation Recombinant saccharomyces
EP0357391A2 (fr) * 1988-08-31 1990-03-07 Allelix Biopharmaceuticals Inc. Excrétion de protéines hétérologues de E. Coli

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AUST. J. BIOL. SCI., Vol. 35, pages 163-72, (1982), MOORE et al. *
J. BIOTECH., Vol. 10, pages 151-160; OHGAI et al.: "Production of Rat Epidermal Growth by E. Coli Cells Containing a Secretion Plasmid", (in Total). *
J. BIOTECH., Vol. 8, pages 77-86, (1988); FUJIMO et al.; "Expression and Secretion of Human Epidermal Growth Factor by E. Coli Using Enterotoxin Signal Sequences", (in Total). *
PROC. NATL. ACAD. SCI. (USA), Vol. 82, pages 7212-7216, (November 1985), OKA et al.; "Synthesis and Secretion of Human Epidermal Growth Factor by E. Coli", (in Total). *

Also Published As

Publication number Publication date
CN1051198A (zh) 1991-05-08
ZA908042B (en) 1991-08-28

Similar Documents

Publication Publication Date Title
US5304473A (en) A-C-B proinsulin, method of manufacturing and using same, and intermediates in insulin production
CA2033176C (fr) Proteines de fusion d'hormones de croissance
JP2566933B2 (ja) 真核細胞融合タンパク質、その生産及び用途、並びに方法
NZ207924A (en) Production of "mature" (191 amino acid residues) human growth hormone from recombinant prokaryotic host
NZ199391A (en) Chimeric polypeptides comprising a proinsulin sequence,and preparation by recombinant dna technique;production of human insulin
AU4663189A (en) Somatotropin analogs
JPH0797995B2 (ja) ペプチド類の製造法
JPH08301899A (ja) Ｉｇｆ−１スーパーアゴニスト
KR100554490B1 (ko) 분자내 샤퍼론 유사 서열을 함유하는 키메라 단백질 및이것의 인슐린 제조용 용도
Hwang et al. A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli
JPH10507452A (ja) ケラチノサイト成長因子の精製法
AU648272B2 (en) Recombinant growth factors
WO1991005863A1 (fr) Facteurs de croissance recombinants
JPH02484A (ja) 突然変異体の酸性繊維芽細胞成長因子
AU2238792A (en) Production and recovery of recombinant neurotrophins
Barthelemy et al. Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase
JPH02503144A (ja) ウシインタ‐ロイキン‐１β
US6531134B1 (en) Mammalian milk growth factor
JP2602628B2 (ja) Ｓｍｃ様ポリペプチド及びその産生方法
RU2143492C1 (ru) Рекомбинантная плазмида, кодирующая гибридный белок - предшественник инсулина человека (варианты), штамм бактерий e.coli - продуцент гибридного белка - предшественника инсулина человека (варианты), способ получения инсулина человека
WO1997000886A1 (fr) Intermediaires de proteines contre l'obesite, leur preparation et leur utilisation
RU2144082C1 (ru) Рекомбинантная плазмида, кодирующая гибридный белок-предшественник инсулина человека (варианты), штамм бактерий e.coli - продуцент гибридного белка-предшественника инсулина человека (варианты) и способ получения инсулина человека
AU730935B2 (en) Bovine milk growth factor
JP2682738B2 (ja) 成長ホルモン融合蛋白質
Smith et al. Production and biological activity of hybrid growth hormone-releasing hormone propeptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH DE DK ES FI GB HU JP KP KR LK LU MC MG MW NL NO RO SD SE SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB IT LU ML MR NL SE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA