EP0425580A1 - Gene de protease de vih synthetique et son procede d'expression - Google Patents

Gene de protease de vih synthetique et son procede d'expression

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Publication number
EP0425580A1
EP0425580A1 EP89909316A EP89909316A EP0425580A1 EP 0425580 A1 EP0425580 A1 EP 0425580A1 EP 89909316 A EP89909316 A EP 89909316A EP 89909316 A EP89909316 A EP 89909316A EP 0425580 A1 EP0425580 A1 EP 0425580A1
Authority
EP
European Patent Office
Prior art keywords
protease
gene
hiv
expression
synthetic
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP89909316A
Other languages
German (de)
English (en)
Other versions
EP0425580A4 (en
Inventor
John M. Louis
Stephen Oroszlan
Peter T. Mora
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
United States, REPRESENTED BY SE
Original Assignee
United States Department of Commerce
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 United States Department of Commerce filed Critical United States Department of Commerce
Publication of EP0425580A1 publication Critical patent/EP0425580A1/fr
Publication of EP0425580A4 publication Critical patent/EP0425580A4/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to synthetic genes and their expression products. Specifically, this
  • invention relates to a synthetic protease gene and its expression product.
  • protease protein in purified virion preparation was shown only by immunological techniques.
  • the HIV protease sequence together with the gag and pol sequence or fusion proteins have been expressed from viral DNA in bacteria. Examples of such disclosures include: 1. Henderson, et al., 1988, "Human Retroviruses, Cancer and AIDS: Approaches to Prevention and Therapy", D. Boloqnesi Ed. Published by Alan R. Liss Inc., New York, NY. pp.135-147;
  • the primary sequences of the HIV protease has been determined by protein analysis and by the
  • protease is a 99 amino acid long protein encoded by a 297bp long stretch of the HIV provirus. All previous experiments on the protease gene and on its expression were carried out by
  • nucleotide sequences cloned out from the cDNA of the provirus The inventors' work using synthetic DNA proves that the nucleotide sequence of the provirus DNA and also the deduced aminoacid sequence are
  • the sequence coding for the protease in the pol open reading frame of HIV was determined by previous analysis and corresponds to nucleotide 1609 to 1906 The N terminus and the C terminal amino-acids are proline and phenylalanine respectively.
  • This sequence coding for the HIV-I 99 aminoacid protease is 297bp long as follows.
  • HIV human immunodeficiency virus
  • the invention is a gene for encoding a protease of human immunodeficiency virus.
  • the gene consists essentially of a synthetic nucleotide sequence for a protease essential to infectivity of human immunodeficiency virus.
  • the protease is desirably a protease of HIV-1 or HIV-2 that is essential for the infectivity of these viruses.
  • HIV-1 protease expression of the HIV-1 protease is represented above by the top rows of nucleotide sequence.
  • Figure 1 presents the expressed HIV protease as analyzed in Western blot.
  • Figure 2 illustrates a strategy for the synthesis of the HIV-1 protease gene.
  • the 3' overhangs are in lower case.
  • the complementary strands (not shown) were provided with 3' overhangs to match the coding strands.
  • Figure 3 illustrates the induction of the gene at various periods of time.
  • Figure 4 illustrates the activity of the expressed protease using a synthetic peptide asa substrate.
  • the invention is a synthetic DNA sequence for encoding a specific enzyme or protease.
  • the protease is essential for the infectivity of the human
  • HIV immunodeficiency virus
  • the invention also includes synthesis and expression of the protease gene of other retroviruses such as HIV-2, the human leukemia viruses such as HTLV I, II, and other human and animal RNA containing viruses causing leukemia sarcoma and other malignencies.
  • nucleotide sequence for the preferred embodiment of this invention was obtained from a published paper by Ratner, et al., supra.
  • the sequence in the pol open reading frame coding for the protease of HIV-1 corresponds to nucleotide 1609 to 1906.
  • the N-terminal and the C-terminal amino-acids are proline and phenylalanine respectively.
  • This sequence coding for the 99 aminoacid protease is 297bp long as shown above. Minor substitutions of one or more bases in this and other genes useful in this invention can produce a variant gene capable of expressing the desired
  • a procaryotic expression vector was used to clone and then to express the synthetic sequence coding for the protease.
  • the expression can be in prokaryotes (bacteria) or in other appropriate expression systems.
  • Recombinant clones screened by colony hybridization using a labelled fragment (62bp) spanning the internal region of the protease gene. Positive clones were further analyzed for the size of the insert. Clones which answered positive were induced for expression and analyzed in Western blots to determine the protein product using specific antibodies.
  • Figure 1 gives an example.
  • the gene product has specific protease activity, as it is capable of cleaving both synthetic and natural substrates.
  • the enzyme has been purified by specific column chromatographic techniques, including affinity chromotography.
  • the method of this invention can produce enough active protease to study the structure of the protease, its mechanisms of
  • Figure 1 demonstrates the expression of the HIV protease in E. coli.
  • Cells transformed with the synthetic sequence of HIV protease in an appropriate expression vector were induced and the bacterial lysate was electrophoresed in SDS-PAGE. After transfer of proteins into a nitrocellulose membrane, immunoblotting procedure was performed using the specific antibody to the HIV protease. Detection of Ag-Ab complex was made using I 125 protein A. The autoradiograph lane A
  • the 11.5 kd band is the protease.
  • the synthetic DNA of the invention also obviates any need to manipulate (infectious) viral material and overcomes limitations in the quantities which can be obtained by other means.
  • Plasmid PKK233-2 a procaryotic expression vector was purchased from Pharmacia. PKK233-2 was used to transform in a laq-q host, E. coli cell JM105 or RB791. The cells were selected in M9 minimal media containing lug/ml thiamine, prior to using them for transformation. All chemicals utilized in the
  • oligonucleotides were from Applied Biosystems Inc. T4 polynucleotide kinase, DNA ligase, and Klenow fragment of E. coli DNA polymerase I were obtained from New England Biolabs. Restriction
  • DNA fragments were synthesized using a ABI DNA synthesizer (model 381A). All synthetic fragments were purified by electrophoresis in a 12%
  • the polyclonal antibodies were raised in rabbits against (i) a complete synthetic sequence of 1 to 99 aminoacids of the HIV-1 protease and (ii) a tridecapeptide corresponding to the C-terminus of the protease.
  • E. coli cells bearing the appropriate plasmid construct were grown to log phase, induced, and lysed by sonication. Total cell extracts were analysed by NaDodSO 4 /PAGE and subjected to immunoblot analysis.
  • Oligopeptides were synthesized in a Peptide Synthesizer (Applied Biosystems Model 430A), according to the method previously published (Copeland and
  • This example represents the preferred embodiment.
  • the nucleotide sequence of the protease gene was taken from Ratner et al.
  • the sequence in the pol open reading frame for the protease gene starts at nucleotide 1609 and ends at 1906, for coding 99 aminoacids. This sequence and its complement were synthesized as five individual fragments of
  • Figure 3 shows examples of Western blot analysis of the gene product.
  • Figure 3 illustrates expression of the synthetic protease gene in E. coli. Clone PR-C bearing the coding sequence to the protease was induced for expression. The proteins (75ug of bacterial extract) were electrophoresed in a NaDodSO 4 /PAGE transferred to nitrocellulose and subjected to immunoblot analysis using a mixture of the two protease specific rabbit polyclonal antibodies raised against (i) a complete synthetic sequence of 1-99 amino acids of the HIV-1 protease and (ii) a tridecapeptide corresponding to the C terminus of the protease.
  • Figure 3A shows the induction of the gene with 0.4mM IPTG at various periods of time.
  • Figure 3B shows the induction for 30 minutes.
  • FIG. 1-5 represent mM concentration of IPTG at 0.28, 0.56, 1.12, 2.24 and 4.48 respectively.
  • Figure 3C shows the analysis after 60 minutes of induction with ImM IPTG and lysing the cells in various buffers.
  • B1 denotes lysis of cells in 50mM Tris-HCl at pH 7.0, 150mM NaCl, ImM EDTA, ImM PMST, ImM DTT and 0.5 percent NP-40.
  • B2 is the same as B1, but without NaCl and
  • B3 is in 50mM potassium phosphate at pH 6.0, ImM PMSF and ImM DTT.
  • B4 is the same as B3 with a pH of 6.5. Positions of protein molecular weight markers are inducated on the left in kilodaltons.
  • E. coli cells bearing plasmid PR-C were grown in Luria broth to an optical density of 0.4 A600nm, and then induced at various periods of time for expression from the trc promotor by adding IPTG (isopropyl-beta-D-thiog-alactopyranoside) at a concentration of 0.4mM as seen in Figure 3A.
  • IPTG isopropyl-beta-D-thiog-alactopyranoside
  • the cloned gene expressed a single, unfused protein band of 11.5kd. Expression was maximal after 30 minutes of induction. This level decreased to about 25 percent at 60 minutes. There was no
  • Figure 4 illustrates the activity of the expressed protease using a synthetic peptide as a substrate. Protease assays were carried out with
  • reaction buffer was mixed with aliquots of various cell extracts (see description of Figure 4 above) and incubated at 37°C. Equal eliquots of incubation mixture were taken at various time points and analyzed by RP-HPLC. The substrate in the 0 hour sample eluted as a single peak as shown in Figure 4A. After incubation for 1 hour, two newly appearing peaks, products
  • Pro-Ile-Val-Glu-NH 2 is substantially smaller than that of the pentapeptide having a free
  • the amino acid composition data for the substrate and its cleavage products are shown in Table 1.
  • the amounts of observed amino acids correspond clearly to the expected amounts demonstrating that the cleavage occurs at the expected cleavage site of the synthetic peptide corresponding to the pl7-p24 site of the gag precursor.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention est une séquence d'ADN synthétique permettant d'encoder une enzyme ou une protéase spécifique. La protéase est essentielle pour l'achèvement (réplication) d'un virus d'immunodéficience humaine (VIH) infectieux. Le gène inventé est utile pour l'expression de la protéase par méthodologie recombinante dans des cellules procaryotiques et/ou eucaryotiques, et pour la production d'une quantité commercialement intéressante de la protéase pour la caractérisation biochimique et physique, nécessaire pour trouver un inhibiteur efficace de la protéase, et ainsi pour bloquer la production de virus d'immunodéficience humaine (VIH) infectieux.
EP19890909316 1988-07-13 1989-07-13 Synthetic hiv protease gene and method for its expression Pending EP0425580A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21830488A 1988-07-13 1988-07-13
US218304 1988-07-13

Publications (2)

Publication Number Publication Date
EP0425580A1 true EP0425580A1 (fr) 1991-05-08
EP0425580A4 EP0425580A4 (en) 1992-05-06

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP19890909316 Pending EP0425580A4 (en) 1988-07-13 1989-07-13 Synthetic hiv protease gene and method for its expression

Country Status (6)

Country Link
EP (1) EP0425580A4 (fr)
JP (1) JPH04500602A (fr)
AU (1) AU635216B2 (fr)
CA (1) CA1340748C (fr)
IL (1) IL90947A (fr)
WO (1) WO1990000556A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE199498T1 (de) * 1991-12-24 2001-03-15 Amrad Corp Ltd Verwendung von leukämieinhibierendem faktor (lif) zur behandlung von tumoren und sarkomen
US6071895A (en) * 1992-03-11 2000-06-06 Narhex Limited Polar-substituted hydrocarbons
JPH07504654A (ja) * 1992-03-11 1995-05-25 ナルヘックス　リミテッド オキソ及びヒドロキシ置換炭化水素のアミン誘導体
MXPA93002392A (es) 1992-03-11 2005-02-04 Narhex Ltd Derivados amino de hidrocarburos-oxo e hidroxi-substituidos.
US5888992A (en) * 1992-03-11 1999-03-30 Narhex Limited Polar substituted hydrocarbons
US6001558A (en) * 1997-06-25 1999-12-14 Ortho Clinical Diagnostics, Inc. Amplification and detection of HIV-1 and/or HIV 2
ES2299276T3 (es) 1998-12-31 2008-05-16 Novartis Vaccines And Diagnostics, Inc. Polipeptidos env del vih modificados.
US7935805B1 (en) 1998-12-31 2011-05-03 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof
AU2487300A (en) 1998-12-31 2000-07-31 Chiron Corporation Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
WO2000039302A2 (fr) 1998-12-31 2000-07-06 Chiron Corporation Expression amelioree de polypeptides hiv et production de particules de type viral
CA2458995C (fr) 2001-08-31 2013-04-30 Chiron Corporation Polynucleotides codant des polypeptides de type b du vih antigeniques, polypeptides et leurs utilisations

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252477A (en) * 1987-06-01 1993-10-12 The United States Of America As Represented By The United States Department Of Health And Human Services Human immunodeficiency virus specific proteolytic enzyme and a method for its synthesis and renaturation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9000556A1 *

Also Published As

Publication number Publication date
CA1340748C (fr) 1999-09-14
EP0425580A4 (en) 1992-05-06
AU635216B2 (en) 1993-03-18
IL90947A (en) 1997-06-10
JPH04500602A (ja) 1992-02-06
WO1990000556A1 (fr) 1990-01-25
IL90947A0 (en) 1990-02-09
AU4058589A (en) 1990-02-05

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