+

WO1990012031A1 - CD4 SOLUBLE PRODUITE DANS $i(E. COLI) - Google Patents

CD4 SOLUBLE PRODUITE DANS $i(E. COLI) Download PDF

Info

Publication number
WO1990012031A1
WO1990012031A1 PCT/US1990/001367 US9001367W WO9012031A1 WO 1990012031 A1 WO1990012031 A1 WO 1990012031A1 US 9001367 W US9001367 W US 9001367W WO 9012031 A1 WO9012031 A1 WO 9012031A1
Authority
WO
WIPO (PCT)
Prior art keywords
scd4
coli
cells
hiv
soluble
Prior art date
Application number
PCT/US1990/001367
Other languages
English (en)
Inventor
Robert L. Garlick
Richard J. Kirschner
William Gary Tarpley
Che-Shen C. Tomich
Original Assignee
The Upjohn Company
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 The Upjohn Company filed Critical The Upjohn Company
Publication of WO1990012031A1 publication Critical patent/WO1990012031A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is in the field of bioprocess engineering. Particularly, the invention relates to soluble CD4 polypeptides produced in E. coli hosts, and to processes for producing such CD4 polypeptides in such hosts. BACKGROUND OF THE INVENTION
  • CD4 or T4 is a 55,000 molecular weight membrane protein found on T4 helper/inducer lymphocytes and is thought to be instrumental in class II major histocompatibility complex (MHC) interactions (S. Meuer, et al, Proc. Natl. Acad. Sci. U.S.A., 798, pp. 4395-99 (1982); W. Biddison, et al, J. Exp. Med. , 156, pp. 1065-76 (1982); Y. Thomas, et al, Immun. Rev., 74, pp. 113-28 (1985); D. Gay, et al, Nature, 328, pp. 763-66 (1987); B.D.
  • MHC major histocompatibility complex
  • CD4 also is the receptor for the gpl20 envelope glyco- protein of human immunodeficiency virus (HIV) , which is the RNA virus responsible for acquired immune deficiency syndrome (AIDS) in humans (D. Klatman, et al, Science, 225, pp. 59-63 (1984). Binding HIV gpl20 protein to CD4 is essential for entry of the virus into the lymphocyte, and subsequent viral proliferation and eventual death of the infected lymphocyte (J. Sodroski, et al, Nature, 322, pp. 470-74 (1986); J. ifson, et al, Nature, 323, pp.
  • HIV human immunodeficiency virus
  • g ⁇ l20 binding to CD4 also is instrumental in the resulting fusion of infected cells with uninfected cells, producing giant multinucleated cells. This cell fusion, or syncytium formation, is a major cytopathic effect induced by HIV (J. Lifson, et al, Science, 232, p. 1123 (1986); B. Yoffe, et al, Proc. Natl. Acad. Sci. U.S.A., 84, p. 1429 (1987)). As the T4 lymphocytes are destroyed by HIV, the patient's immune system becomes compromised, and the patient is more prone to contracting adventitious diseases and infections.
  • CD4 or a portion of the CD4 molecule can bind gpl20, can block the infection of T4 lymphocytes with HIV in vitro, and can inhibit the associated cell fusion or syncytium formation associated with HIV infection (Yoffe, B., et al., Proc. Natl. Acad. Sci. U.S.A., 84, 1429 (1987); Weiss, R.A., Nature, 331, 15 (1988); Smith, D.H., et al, Science, 238, 1704- 1707 (1987); Fisher, R.A.
  • CD4 could sequester free gpl20 shed by HIV-infected lymphocytes or mask gpl20 that may have bound to uninfected cells (Smith, D.H. , et al, supra) since uninfected lymphocytes that have bound gpl20 become targets for antibody-dependent cellular toxicity (Lyerly, H. , et al, Proc. Natl. Acad. Sci. U.S.A., 84, 4601 (1987)).
  • the intact or precursor CD4 molecule contains a signal peptide, a 370-amino acid extracellular region containing four domains, a membrane spanning domain, and an intracellular domain containing 40 amino acid residues (Maddon, P., et al, Cell, 42, 93 (1985); Clark, S., et al, Proc. Natl. Acad. Sci. U.S.A., 84, 1649 (1987)).
  • Lasky and coworkers (Lasky, et al, Cell, 50, 975 (1987)) expressed the entire CD4 protein in Chinese hamster ovary cells. The CD4 expressed by these cells migrated to the cell membrane and would bind gpl20 in an in vitro experiment.
  • soluble CD4 constructs containing 364, 374, and 377 amino acid residues (essentially the extracellular four domain region of CD4) could be expressed and secreted by CHO cells into the growth medium, would successfully bind gpl20, and would block HIV infection and syncytial formation in vitro.
  • sCD4 sCD4 molecule of unknown chain length from recom- binant CHO cells. Their molecule also bound gpllO (sic), and inhibited the binding of HIV to the lymphocytes, resulting in inhibition of virus infectivity. This group also reported expressing a sCD4 molecule (also of unknown length) as a denatured or non- native protein in bacteria. No mention was made of attempts to renature the sCD4 protein from bacteria.
  • the present invention relates to soluble CD4 the produced in E. coll .
  • the soluble CD4 produced in E. coli consists of the first N-terminal two domains of CD4, more particularly, the first 183 N-terminal amino acids of CD4.
  • soluble CD4 or sCD4 refers to the about 370-amino acid extracellular portion of the CD4 molecule comprising essentially the amino terminal four domains.
  • the first two domains of sCD4 refers to the amino-terminal portion of the CD4 polypeptide consisting of about 180 amino acids, as it is understood in the art, and closely related derivatives thereof that also bind gpl20.
  • CD4 polypeptide and amino acid sequence of CD4 are known to those skilled in the art as set forth in certain of the documents cited above which are hereby incorporated by reference.
  • Ve have cloned a DNA sequence encoding a sCD4 molecule contain ⁇ ing 183 amino acid residues (sCD4-183), i.e., the first two domains of the mature CD4 molecule, in E. coli .
  • sCD4-183 183 amino acid residues
  • Culture development programs were applied to E. coli D112 and K12S transformed with the pBR322-related vectors, as well as a pURA transformant in E. coli BST-lc, and isolates with desirable pheno- types were obtained.
  • a selection/passaging procedure yielded a superior culture for producing sCD4-183 containing a pBR322-based vector (pUC 1391), contained in an E. coli strain designated KU141 (UC 12420) .
  • This strain (including the vector) was deposited at the Agricultural Research Service Culture Collection (NRRL), Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A., on 28 March 1989 in accordance with the requirements of the Budapest Treaty and was given accession number NRRL B-18474 .
  • the protein is expressed as insoluble inclusion bodies at the 1-2 gram- per-liter level in 10- and 200-liter fermentations, and is success ⁇ fully refolded and purified.
  • EXAMPLE 1 Construction of a Plasmid for High Level Expression of CD4-183 in E. coli Isolation of DNA fragments, preparation of plasmids, use of restriction endonucleases and other DNA modifying enzymes, transfor ⁇ mation of E. coli , and other cloning techniques are done according to procedures described in the Molecular Cloning Manual by T. Maniatis, E.F. Fritsch and J. Sambrook, Cold Spring Harbor Laboratory (1982) which is incorporated herein by reference. To express sCD4-183, the DNA sequence coding for this region is isolated from either plasmid pSP65-T4 (P. Maddon, et al, Cell, 42, pp. 93-104 (1985); obtained from Dan R.
  • Plasmid pAc373/T4ex is obtained from Ellis L. Reinherz, Harvard Medical School and was derived from pSP65- T4 (R.E. Hussey et al, Nature, 331, pp. 78-81 (1988)).
  • the 1.1 kb Rsal-BamHI fragment coding for amino acid residues 17-369 of CD4 is isolated from pAc373/T4ex.
  • This Rsal-BamHI fragment is ligated to the expression vector pTrp2 treated with Clal and BamHI, together with 2 oligonucleotides which provide codons for residues 1-16 of CD4, the ATG initiation codon, and the ends cor ⁇ responding to Clal and Rsal restriction sites.
  • the oligonucleotides are 5' CGATAATGAAAAAGGTAGTTCTGGGTAAAAAGGGCGATACCGTGGAACTGACTTGT and its complementary strand; oligonucleotide synthesis and purification are according to the procedures described in M.K.
  • pTrp2-CD-4 The resulting plasmid, pTrp2-CD-4, is analyzed by restriction endonucleases and sequenced to verify the presence of the beginning of the sequence encoding CD4.
  • the DNA sequencing procedure is a modification of that described by R.B. Wallace, et al, Gene, 16, pp. 21-26 (1981).
  • Expression vector pTrp2 contains the promo ⁇ ter/operator region of the E. coli tryptophan synthetic pathway and a riboso e binding site rich in A and T bases to enhance translation initiation (M.K. Olsen, et al, supra).
  • plasmid pTrp2-CD4/Kpn is made.
  • the large BamHI-Aval fragment is isolated from pTrp2-CD4 and ligated to 2 oligonucleotides (5' TCGGGTAC- CTAGCTAAG and its complementary strand) which carry Aval sticky ends at the 5' end followed by a Kpnl site, 2 translation stop codons and BamHI sticky ends.
  • the resulting plasmid, pTrp2-CD4 Kpn is treated with Kpnl, Klenow enzyme, and Stul, and the large fragment is isolated.
  • pTrp2-CD4/183 (pUC 1391)
  • the CD4 encoding sequence is under the control of the E. coli tryptophan promoter and a riboso e binding site rich in A and T bases in a pBR background.
  • the product of this expression vector has 184 amino acid residues, beginning with et- lys-lys-val-val-leu and ending with phe-gln-lys-ala-ser.
  • EXAMPLE 2 E. coli strain for Producing Soluble CD4
  • E. coli strain KU141 UC 12420
  • a passaged transformant of E. coli K12S containing plasmid pTrp2-CD4/183 pUC 1391
  • EXAMPLE 3 Preparations of Soluble CD4 by E. coli in Shake Flasks
  • the expression culture KU141 is grown at 27*C in S584 (IX RJK 584 salts (pH 6.8), 1% yeast extract, 1 mM MgS0 4 , 0.8% glycerol, supplemented with 50 ⁇ g/ml ampicillin;
  • IX RJK584 salts contains K 2 HP0 4 (2.6 g 1) , NaNH HP0 4 -4H 2 0 (10.6 g/1) , citric acid (2.0 g/1), and (NH 4 ) 2 S0 4 (0.66 g/1)) to 5-6 A 550 and diluted into M584C (a minimal medium supplemented with amino acids;
  • EXAMPLE 4 E. coli Fermentations at the 10, 200 and 5000 Liter Scales A preferred procedure for fermentations allows the KU141 cells to grow at 27* where plasmid stability is better, and after shifting 10 to 37* , express product under conditions supporting highest specific activity and titer. No expression is detected in the harvest sample from seed tanks containing S584 medium and cells at 5-6 A550. The expression tanks containing medium M584C are inoculated at 0.25 A55 Q and shifted to 37 ⁇ C at a higher cell density (e.g., 1.5-6 A550). 15 Ampicillin shots (40 g/200 1) are delivered at various times, e.g., at inoculation, temperature shift, and 5-6 hours post shift.
  • the fermentors are prepared with 1.25% glucose, the cultures achieve a maximum density of * s35 550 at 20 hours.
  • a constant specific activity of 40 g product/A.1 is attained a few hours after the 20 temperature shift with final titers of 1.4 g/1.
  • a preferred method for solubilizing SCD4-183 is to add to a cell pellet (lA'ml of E. coli expressing the product) 200 ⁇ l of 50 mM ethanolamine-HCl, pH 10-10.5, 2% SDS, 1.5% mercaptoethanol, 5% glycerol, 0.01% bromphenol blue and boil for 2-5 min.
  • a preferred method to minimize problems with DNA-related viscosity is first to add to the cell pellet 150 ⁇ l of 25 mM ethanolamine, pH 10-10.5, 0.1 mM MgCl 2 , 0.2% SDS, and a relatively stable nuclease, e.g., 30 units/ml Benzon nuclease; DNA is partially degraded during an incubation, e.g., 30 minutes at room temperature. Then, add 50 ⁇ l of an appropriate electrophoresis sample buffer, e.g., 200 mM ethanola ⁇ mine-HCl, pH 10-10.5, 8% SDS, 6% mercaptoethanol, 20% glycerol, 0.05% bromphenol blue, and boil for 2-5 min. Such samples could be loaded directly onto SDS-PAGE gels.
  • the aggregated, denatured sCD4-183 made in E. coli did not solubilize well or reproducibly for preparative purposes under conditions effective with some other recombinant proteins but did when chaotropic agents were used at alkaline pH (9.5-11). Fermenta- tion beer samples were harvested, and the cells were killed with toluene, preferably using >20 ml toluene/liter of beer for cell densities of ⁇ 50 A550. The cells were then centrifuged at 10,000 RPM at 4* C for 30_ minutes. The cells could be lysed enzymatically in the following manner: to the centrifuged cells from one liter of beer were added 190 ml of 0.02 M Tris-HCl buffer, pH 8.0, containing
  • Tergitol 15-S-7 were added, and the sample was vigorously mixed for another 30 minutes.
  • the cells could be disrupted mechanically using a device such as a French Press or a Gaulin M3
  • the lysed cells were centrifuged, e.g., for 30 minutes at 10,000 rpm and 4* C, and the inclusion body pellet was washed three times, e.g., in 0.02 M Tris-HCl buffer at 4'C.
  • the precipitated and washed inclusion bodies were treated with chaotropic agents to promote solubilization, including 8 M urea or 6 M guanidine-HCl.
  • the inclusion bodies were dis ⁇ solved in 8 M urea plus 0.1 M ethanola ine, pH 10.0, at an approxim- ate protein concentration of 5-20 mg/ml.
  • the sample was then diluted 10-40-fold in 3.5 M urea, 0.1 M ethanolamine, pH 10.0.
  • the sample was stirred vigorously overnight at room temperature, after which the denaturant was removed by dialysis against 0.02 M Tris-HCl, pH 6.8.
  • the inclusion body sample which was 6 M in guanidine-HCl was diluted 10-40-fold in 2 M guanidine-HCl, 0.1 M in ethanolamine, pH 10.0, after which it was stirred vigorously overnight.
  • the sample which was in 0.02 M Tris-HCl, pH 6.8, was applied to a 2.5 X 4.5 cm column of a monoclo ⁇ nal antibody affinity chromatographic column using a monoclonal antibody which binds to the first or N-terminal domain of CD4.
  • the monoclonal antibody was bound to Affi-Gel 10, a commercial resin from BioRad which forms covalent bonds between amino groups (e.g., on proteins or low molecular weight capping reagents) and the resin's N- hydroxysuccini ide ester.
  • the column was washed with 0.02 M Tris-HCl, pH 6.8, until the 280 nm absorbance of the eluant returned to zero.
  • the sCD4-183 then was eluted with 0.1 M glycine-HCl, pH 2.5, concentrated approximately 10-fold, e.g., by use of a Centricon centrifugal concentrator (Amicon) , and subjected to gel filtration chromato- graphy, e.g., on a 2.5 X 50 cm column of BioGel P-100 (BioRad) in 0.1 M ammonium bicarbonate buffer, pH 7.8.
  • the sCD4-183 was again con ⁇ centrated, followed by freezing at -80 ⁇ C.
  • Physicochemical analyses - of the sCD4-183 indicate that it is homogeneous on SDS-PAGE after staining with silver, has one N- terminal amino acid sequence, begins with methionine (Met-Lys-Lys- etc), and has an amino acid composition closely matching the theoretical. That the purified sCD4-183 binds purified gpl20 in solution was determined by a binding assay which quantifies the competition of SCD4-183 with sCD4-369 for g ⁇ l20. For example, preincubation of gpl20 for 15 minutes with an equal molar amount of SCD4-183 inhibits sCD4-369 binding by 70%. sCD4-182 (CHO) exhibits similar inhibition in this assay.
  • EXAMPLE 7 Biological Activity of sCD4 Made in E. coli sCD4-183 made in E. coli according to the Examples above, sCD4- 369 consisting essentially of the N-terminal four domains of CD4 and made in Chinese Hamster Ovary (CHO) cells, and sCD4-T4exl consisting essentially of the N-terminal four domains of CD4 and made using a baculovirus expression system known in the art (Hussey, et al, supra) were tested in two different anti-HIV assays: 1) syncytia formation in Jurkat cells " transfected with a gpl20-expression vector and, 2) inhibition of HIV infection of Jurkat cells as determined by reverse transcriptase (RT) enzymatic activity 5 and 8 days post-infection, (some of these cells were also examined for HIV protein synthesis by radioimmune precipitation (RIP) with AIDS patients' sera).
  • RT reverse transcriptase
  • SDC4-183, SCD4-369, and sCD4T4exl are potent anti-HIV agents.
  • concentration of sCD4 used in the infectivity experiments there is no evidence of HIV replication by RT assay 8 days after infection.
  • RIP data indicate that these sCD4s significantly inhibit HIV protein synthesis 3 days post-infection of Jurkat cells.
  • a syncytia assay also compared the sCD4s in a dose-response fashion.
  • sCD4-183 made in E. coli completely blocked syncytia formation while cultures with sCD4-369 made in CHO cells had 100% syncytia.
  • sCD4-369 Even at about 1.4 ⁇ g of SCD4-369, the cultures were not fully protected (i.e., 50% syncytia formation).
  • the IC50s estimated from this experiment are about 0.025 ⁇ g/ml and 1.4 ⁇ g/ml for sCD4-183 and sCD4-369, respectively.
  • sCD4-183 When the levels of HIV RNA are quantified the results are similar. At 0.25 ⁇ g/ml of sCD4-183 there is 89 and 94% inhibition of HIV RNA levels compared to controls 3 and 4 days post-infection (95.5, 111.8 pg RNA/ml compared to control values of 891 and 1756). sCD4-369 made in CHO cells at 0.25 ⁇ g/ml did not inhibit HIV RNA synthesis at these times. Because 10-fold dilutions of the sCD4s were used in these experiments the IC50s cannot be accurately calculated. However, they are estimated to be 25-250 ng/ml for sCD4- 183 and 250-2500 ng/ml for sCD4-369.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des polypeptides de CD4 soluble produits dans E. Coli, présentant une activité améliorée. L'invention concerne également des procédés de fabrication de polypeptides de CD4 soluble.
PCT/US1990/001367 1989-04-05 1990-03-20 CD4 SOLUBLE PRODUITE DANS $i(E. COLI) WO1990012031A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33351689A 1989-04-05 1989-04-05
US333,516 1989-04-05

Publications (1)

Publication Number Publication Date
WO1990012031A1 true WO1990012031A1 (fr) 1990-10-18

Family

ID=23303119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/001367 WO1990012031A1 (fr) 1989-04-05 1990-03-20 CD4 SOLUBLE PRODUITE DANS $i(E. COLI)

Country Status (2)

Country Link
AU (1) AU5437190A (fr)
WO (1) WO1990012031A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001304A1 (fr) * 1986-08-21 1988-02-25 The Trustees Of Columbia University In The City Of Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida
WO1989001940A1 (fr) * 1987-09-04 1989-03-09 Biogen, Inc. Sequences d'adn, molecules d'adn recombinant et procedes de production de proteines t4 solubles
EP0331356A2 (fr) * 1988-02-24 1989-09-06 Smithkline Beckman Corporation Expression de protéines de fixant au VIH

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001304A1 (fr) * 1986-08-21 1988-02-25 The Trustees Of Columbia University In The City Of Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida
WO1989001940A1 (fr) * 1987-09-04 1989-03-09 Biogen, Inc. Sequences d'adn, molecules d'adn recombinant et procedes de production de proteines t4 solubles
EP0331356A2 (fr) * 1988-02-24 1989-09-06 Smithkline Beckman Corporation Expression de protéines de fixant au VIH

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nature, Volume 335, No. 6188, 22 September 1988, V.K. CHAUDHARY et al.: "Selective Killing of HIV-Infected Cells by Recombinant Human CD4-Pseudomonas Exotoxin Hybrid Protein", pages 369-372 *

Also Published As

Publication number Publication date
AU5437190A (en) 1990-11-05

Similar Documents

Publication Publication Date Title
CA1341611C (fr) Recepteur de l'interleukine
DE69535495T2 (de) Expression und exporttechnologie von proteinen als an immunoglobin gebundene fusionsproteine
DE3855523T2 (de) Proteine mit Faktor-VIII-Aktivität, Verfahren zu deren Herstellung unter Verwendung von genetisch modifizierten Zellen und diese enthaltende pharmazeutische Zusammensetzungen
EP0255231B1 (fr) Gène humain d'érythropoiétine: expression à haut niveau dans des cellules de mammifères transférées de façon stable
CA1317244C (fr) Production de formes biologiquement actives de csf a l'aide d'un systeme d'expression baculovirus (acnpv) - cellule d'insecte
US5712121A (en) Chimeric interleukin 5-receptor/immunoglobulin polypeptides
EP0623674A1 (fr) Récepteurs d'interleukine-1
GARLICK et al. Escherichia coli expression, purification, and biological activity of a truncated soluble CD4
WO1991018983A1 (fr) Proteine recombinante de liaison d'un antigene viral complexe du vih-1
IE61119B1 (en) Nucleic acid encoding tgf-beta and its uses
Ban et al. Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor
EP0693937A1 (fr) Vaccins derives de vih-1, compositions d'anticorps correspondantes et leur utilisation therapeutique et prophylactique
WO1990010070A1 (fr) Analogues d'il-2 contenant des sites de glycosylation a liaison n
NZ226475A (en) Soluble hcd4 (t4) t-lymphocyte surface glycoprotein that binds hiv gp120 envelope protein
AU7372596A (en) Chemokine inhibitor
US5705484A (en) Biologically active polypeptide fusion dimers
EP0577894B1 (fr) Conception, construction et expression de protéines chimériques pour le développement de vaccins et de réactifs diagnostiques
CA2305341C (fr) Stabilisation des glycoproteines trimeres d'enveloppe au moyen de liaisons bisulfure introduites dans un ectodomaine de la glycoproteine gp41
DE69023580T2 (de) Verfahren zur Expression von, als Fusionsproteine produzierten, heterologen Proteinen in E. coli, deren Verwendungen, Expressionsvektoren und rekombinante Stämme.
EP0437427A1 (fr) Dimeres clives de polypeptides semblables a substance d'inhibition mullerienne
CA2000989A1 (fr) Conjugats de proteines t4 solubles et de toxines et methode de traitement pour la prevention de l'infection par les virus du sida, arc & hiv
Chen et al. Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.
WO1990012031A1 (fr) CD4 SOLUBLE PRODUITE DANS $i(E. COLI)
AU702379B2 (en) Novel feline cytokine protein
JPH05103675A (ja) ヒト神経成長因子２の製造法

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 1990906547

Country of ref document: EP

AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA FI HU JP KP KR LK MC MG MW NO RO SD 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

WWW Wipo information: withdrawn in national office

Ref document number: 1990906547

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载