CA1340748C - Synthetic hiv protease gene and method for its expression - Google Patents
Synthetic hiv protease gene and method for its expressionInfo
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- CA1340748C CA1340748C CA000604978A CA604978A CA1340748C CA 1340748 C CA1340748 C CA 1340748C CA 000604978 A CA000604978 A CA 000604978A CA 604978 A CA604978 A CA 604978A CA 1340748 C CA1340748 C CA 1340748C
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- protease
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention is a synthetic DNA sequence for encoding a specific enzyme or protease. The protease is essential for the completion (replication) of an infective human immunodeficiency virus (HIV). The invented gene is desirable for the expression of the protease by recombinant methodology in prokaryotic and/or eukanotic cells and the production of a commercially desirable amount of the protease for biochemical and physical characterization, necessary to find effective inhibitor of the protease, and thereby to block the production of infectious human immunodeficiency virus (HIVs).
Description
..-.~.
2 A:~tD METHOD FOR ITS EXPRESSION
3 'BACKGROUND OF THE INVENTION
4 1. Field of the Invention This invention relates to synthetic genes and their 6 expression products. Specifically, this invention 7 relates to a synthetic protease gene and its expression 8 product. , 9 2. Description of the P,elated Art The presence of protease protein in purified virion 11 preparation was shown only by immunological techniques.
12 The HIV protease sequence together with the gag and pol 13 sequence or fusion proteins have been expressed from 14 viral DLIA in bacteria. Examples of such disclosures include: 1. Henderson, et al., 1988, "Human 16 Retroviruses, Cancer and AIDS: Approaches to Prevention 17 and Therapy", D.. Bolo~nesi Ed. Published by Alan R. Liss 18 Inc., New York, NY. pp.135-147; 2. Debouck, et al., 19 1987, P.N.A.S., 84:8903-8906; and 3. Mous, et al., 1988, J. Virol, 62:1433-1436.
21 The primary sequences of the HIV protease has been 22 determined by protein analysis and by the nucleotide 23 sequence of the proviral DNA. It was thus determined _2-13!0'748 ,_,..., that t'ne protease is a 99 amino acid long protein encoded 2 by a 297bp long stretch of the HiV provirus. All 3 previous experiments on the protease gene and on its 4 expression were carried out by utilizing nucleotide sequences cloned out from the cDLdA of the provirus. The 6 inventors' work using synthetic DMA proves that the 7 nucleotide sequence of the provirus DNA and also the 8 deduced aminoacid sequence are correct.
9 The complete nucleotide sequence of the HIV-1 proviral DNA was published by Rather et al., 1985, 11 Nature, 313:277-284. The sequence coding for the 12 protease in the pol open reading frame of HIV was --13 determined by previous analysis and corresponds to 14 nucleotide 1609 to 1906 The N terminus and the C
terminal amino-acids are proline and phenylalanine 16 respectively. 'This sequence coding for the ~iIV-I 99 17 aminoacid protease is 297bp long as follows.
19 CCTCAGATCA CTCT'TTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAG
GGAGTCTAGT GAGA,AACCGT TGCTGGGGAG CAGTGTTATT iCTATCCCCC CGTTGATTTC
22 GAAGCTCTAT TAGA'TACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
23 CTTCGAGATA ATCT,ATGTCC TCGTCTACTA TGTCATAATC TTCTTTACTC AAACGGTCCT
24 130 1~+0 150 160 170 180 AGATGGAAAC CAAA.~ATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
26 TCTACCTTTG GTTT'TTACTA TCCCCCTTAA CCTCCAAAAT AGTTTCATTC TGTCATACTA
29 GTCTATGAGT ATCT'CTAGAC ACCTGTATTT CGATATCCAT GTCATAATCA TCCTGGATGT
--CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
GGACAGTTGT ATTAACCTTC TTTAGACAAC TGAGTCTAAC CAACGTGAAA TTTAAAA
The industry is lacking a synthetic DNA sequence that encodes a specific enzyme or protease which is essential for the completion replication) of an infective human immunodeficiency virus (HIV). This DNA sequence is desirable to express this protease by recombinant methodology in bacteria and or in eukaryotic cells, and to produce enough protease for biochemical and physical characterization in order to design and produce potent inhibitors of this enzyme, and thereby to block the-production of infective HIV particles.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a recombinant protein having proteolytic activity coded by a nucleotide sequence consisting essentially of:
CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAG
GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
;CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT.
or allelic or species variation thereof or fragments of the nucleotide sequence or the allelic or species variation thereof capable of coding the protein having the proteolytic activity.
Also disclosed are the method for producing this protein and an expression vector having this nucleotide sequence which codes ;'for the protein having proteolytic activity.
3a ~3~d~~~
The invention includes a method of producing a recombinant protein having proteolytic .activity comprising the step of:
(a) introducing into a host cell a nucleotide sequence of:
CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAC
i Pro GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
I
Phe or allelic or species variation thereof, capable of encoding an amino acid sequence having the same amino acid sequence as the protein having the proteolytic activity, (b) culturing the resultant host cells of step a) under conditions such that the sequence or allelic or species variations thereof, is expressed and thereby producing the protein; and (c) isolating the protein.
1340'48 3b There is further provided a construct comprising (a) a DNA gene sequence of CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAC
I
Pro GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
CCTGTCAACA TAATTGGAA.G AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
I
Phe or allelic or species variations thereof capable of encoding the same amino acid sequence as said DNA gene sequence, said sequence having proteolytic activity'; and (b) an expression vector.
Finally, the above construct may also comprise a regulatory sequence operatively linked to the 1~NA gene sequence or allelic or species variation thereof.
D
_ . _ 134074$
,..:
2 Figure 1 presents the expressed HIV protease as 3 analyzed in Western blot.
4 Figure 2 illustrates a strategy for the synthesis of S the HIV-1 protease gene. The 3' overhangs are in lower 6 case. The complementary strands (not shown) were 7 provided with ;i' overhangs to match the coding strands.
8 Figure 3 illustrates the induction of the gene at 9 various periods. of time.
Figure 4 illustrates the activity of the expressed 11 protease using a synthetic peptide asa substrate.
12 DESCRIPTION OF THE PREFERRED EI~IBODII~1ENT
13 The invention is a synthetic DNA sequence for 14 encoding a specific enzyme or protease. The protease is essential for the infectivity of the human 16 immunodeficiency virus (HIV). The invented gene is 17 desirable for the expression of the protease by 18 recombinant methodology in bacteria and or in eukaryotic 19 cells and the production of a commercially desirable amount of the protease for biochemical and physical 21 characterization. This characterization is necessary for 22 the design and production of potent inhibitors of this 23 enzyme. The invention also includes synthesis and 24 expression of the protease gene of other retroviruses such as HIV-2, the human leukemia viruses such as HTLV I, 26 II, and other human and aninal RNA containing viruses 27 causing leukemi<~. sarcoma and other malignencies.
12 The HIV protease sequence together with the gag and pol 13 sequence or fusion proteins have been expressed from 14 viral DLIA in bacteria. Examples of such disclosures include: 1. Henderson, et al., 1988, "Human 16 Retroviruses, Cancer and AIDS: Approaches to Prevention 17 and Therapy", D.. Bolo~nesi Ed. Published by Alan R. Liss 18 Inc., New York, NY. pp.135-147; 2. Debouck, et al., 19 1987, P.N.A.S., 84:8903-8906; and 3. Mous, et al., 1988, J. Virol, 62:1433-1436.
21 The primary sequences of the HIV protease has been 22 determined by protein analysis and by the nucleotide 23 sequence of the proviral DNA. It was thus determined _2-13!0'748 ,_,..., that t'ne protease is a 99 amino acid long protein encoded 2 by a 297bp long stretch of the HiV provirus. All 3 previous experiments on the protease gene and on its 4 expression were carried out by utilizing nucleotide sequences cloned out from the cDLdA of the provirus. The 6 inventors' work using synthetic DMA proves that the 7 nucleotide sequence of the provirus DNA and also the 8 deduced aminoacid sequence are correct.
9 The complete nucleotide sequence of the HIV-1 proviral DNA was published by Rather et al., 1985, 11 Nature, 313:277-284. The sequence coding for the 12 protease in the pol open reading frame of HIV was --13 determined by previous analysis and corresponds to 14 nucleotide 1609 to 1906 The N terminus and the C
terminal amino-acids are proline and phenylalanine 16 respectively. 'This sequence coding for the ~iIV-I 99 17 aminoacid protease is 297bp long as follows.
19 CCTCAGATCA CTCT'TTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAG
GGAGTCTAGT GAGA,AACCGT TGCTGGGGAG CAGTGTTATT iCTATCCCCC CGTTGATTTC
22 GAAGCTCTAT TAGA'TACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
23 CTTCGAGATA ATCT,ATGTCC TCGTCTACTA TGTCATAATC TTCTTTACTC AAACGGTCCT
24 130 1~+0 150 160 170 180 AGATGGAAAC CAAA.~ATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
26 TCTACCTTTG GTTT'TTACTA TCCCCCTTAA CCTCCAAAAT AGTTTCATTC TGTCATACTA
29 GTCTATGAGT ATCT'CTAGAC ACCTGTATTT CGATATCCAT GTCATAATCA TCCTGGATGT
--CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
GGACAGTTGT ATTAACCTTC TTTAGACAAC TGAGTCTAAC CAACGTGAAA TTTAAAA
The industry is lacking a synthetic DNA sequence that encodes a specific enzyme or protease which is essential for the completion replication) of an infective human immunodeficiency virus (HIV). This DNA sequence is desirable to express this protease by recombinant methodology in bacteria and or in eukaryotic cells, and to produce enough protease for biochemical and physical characterization in order to design and produce potent inhibitors of this enzyme, and thereby to block the-production of infective HIV particles.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a recombinant protein having proteolytic activity coded by a nucleotide sequence consisting essentially of:
CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAG
GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
;CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT.
or allelic or species variation thereof or fragments of the nucleotide sequence or the allelic or species variation thereof capable of coding the protein having the proteolytic activity.
Also disclosed are the method for producing this protein and an expression vector having this nucleotide sequence which codes ;'for the protein having proteolytic activity.
3a ~3~d~~~
The invention includes a method of producing a recombinant protein having proteolytic .activity comprising the step of:
(a) introducing into a host cell a nucleotide sequence of:
CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAC
i Pro GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
CCTGTCAACA TAATTGGAAG AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
I
Phe or allelic or species variation thereof, capable of encoding an amino acid sequence having the same amino acid sequence as the protein having the proteolytic activity, (b) culturing the resultant host cells of step a) under conditions such that the sequence or allelic or species variations thereof, is expressed and thereby producing the protein; and (c) isolating the protein.
1340'48 3b There is further provided a construct comprising (a) a DNA gene sequence of CCTCAGATCA CTCTTTGGCA ACGACCCCTC GTCACAATAA AGATAGGGGG GCAACTAAAC
I
Pro GAAGCTCTAT TAGATACAGG AGCAGATGAT ACAGTATTAG AAGAAATGAG TTTGCCAGGA
AGATGGAAAC CAAAAATGAT AGGGGGAATT GGAGGTTTTA TCAAAGTAAG ACAGTATGAT
CAGATACTCA TAGAAATCTG TGGACATAAA GCTATAGGTA CAGTATTAGT AGGACCTACA
CCTGTCAACA TAATTGGAA.G AAATCTGTTG ACTCAGATTG GTTGCACTTT AAATTTT
I
Phe or allelic or species variations thereof capable of encoding the same amino acid sequence as said DNA gene sequence, said sequence having proteolytic activity'; and (b) an expression vector.
Finally, the above construct may also comprise a regulatory sequence operatively linked to the 1~NA gene sequence or allelic or species variation thereof.
D
_ . _ 134074$
,..:
2 Figure 1 presents the expressed HIV protease as 3 analyzed in Western blot.
4 Figure 2 illustrates a strategy for the synthesis of S the HIV-1 protease gene. The 3' overhangs are in lower 6 case. The complementary strands (not shown) were 7 provided with ;i' overhangs to match the coding strands.
8 Figure 3 illustrates the induction of the gene at 9 various periods. of time.
Figure 4 illustrates the activity of the expressed 11 protease using a synthetic peptide asa substrate.
12 DESCRIPTION OF THE PREFERRED EI~IBODII~1ENT
13 The invention is a synthetic DNA sequence for 14 encoding a specific enzyme or protease. The protease is essential for the infectivity of the human 16 immunodeficiency virus (HIV). The invented gene is 17 desirable for the expression of the protease by 18 recombinant methodology in bacteria and or in eukaryotic 19 cells and the production of a commercially desirable amount of the protease for biochemical and physical 21 characterization. This characterization is necessary for 22 the design and production of potent inhibitors of this 23 enzyme. The invention also includes synthesis and 24 expression of the protease gene of other retroviruses such as HIV-2, the human leukemia viruses such as HTLV I, 26 II, and other human and aninal RNA containing viruses 27 causing leukemi<~. sarcoma and other malignencies.
-5- 1340'48 1 The nucleotide sequence for the preferred embodiment 2 of this invention was obtained from a published paper by 3 Ratner, et al., supra. The sequence in the pol open 4 reading frame coding for the protease of HIV-1 corresponds to nucleotide 1609 to 1906. The N-terminal 6 and the C-terminal amino-acids are proline and 7 phenylalanine respectively. This sequence coding for the 8 99 aminoacid protease is 297bp long as shown above.
9 Minor substitutions of one or more bases in this and other genes useful in this invention can produce a 11 variant gene capable of expressing the desired protease.
12 This sequence was synthesized as five fragments~using 13 the DNA synthesizer. Complementary strands corresponding 14 to these five fragments were also synthesized. The 3' overhangs of four bases were provided for appropriate 16 sequences to efficiently ligate each of the five 17 fragments and to provide the correct coding sequence of 18 the protease gene. Nucleotide ATG were added to the 19 fragment corresponding to the 5' end of the gene and TAA
at the 3' end.
21 A procaryotac expression vector was used to clone and 22 then to express the synthetic sequence coding for the 23 protease. The expression can be in prokaryotes 24 (bacteria) or in other appropriate expression systems.
Recombinant clones screened by colony hybridization using 26 a labelled fragment (62bp) spanning the internal region 27 of the protease gene. Positive clones were further 28 analyzed for thE: size of the insert. Clones which 29 answered positi~ae were induced for expression and analyzed in Western blots to determine the protein 31 product using specific antibodies. Figure 1 gives an 32 example.
1 Of the clones screened so far, 3 clones have been 2 identified to express a product of ll.Skd, reacting 3 against specific antibodies as illustrated in Figure 1.
4 Conditions for the induction of a protease gene were S studied in E. coli and optimized. The inventors have 6 shown that the gene product has specific protease 7 activity, as it is capable of cleaving both synthetic and 8 natural substrates. The enzyme has been purified by 9 specific column chromatographic techniques, including affinity chromotography. The method of this invention 11 can produce enough active protease to study the structure 12 of the protease, its mechanisms of action, with a goal of 13 devising specific inhibitors to this enzyme, of a 14 therapeutic application for the treatment of the diseases, such as AIDS, caused by the viruses. Other 16 embodiments of this invention can utilize a gene to 17 express another protease such as the following gene for 18 the HIV-2 protease.
GTAGAAGTCTTGTTAGACACAGGGGCTGACGACTCAATAGTAGCAGGAATAGAGTTA
23 ACAGGCGACACCCAA'TCAACATTTTTGGCAGAAATATTCTGACAGCCTTAGGCATGT
Figure 1 demonstrates the expression of the HIV
26 protease in E. coli. Cells transformed with the 27 synthetic sequence of HIV protease in an appropriate 28 expression vector were induced and the bacterial lysate 29 was electrophor.esed in SDS-PAGE. After transfer of proteins into a nitrocellulose membrane, immunoblotting 31 procedure was performed using the specific antibody to 32 the HIV protease. Detection of Ag-Ab complex was made 33 using 1125 protein A. The autoradiograph lane A
_7_ 1340'~4~
1 represents E. coli transformed with the plasmid, and 2 lanes B and C coli transformed with the plasmid E.
3 bearing syntheticDNA encoding the HIV protease. On the 4 left are protein molecular weight markers in kilodalton.
S The 11.5 kd band is the protease.
6 The synthetic DNA of the invention also obviates any 7 need to manipulate (infectious) viral material and 8 overcomes limitations in the quantities which can be 9 obtained by other means.
EXAMPLES
11 The following materials and methods were used to 12 perform the examples.
13 PLASMID, BACTERIAL STRAINS, AND CHEMICALS:
14 Plasmid PKK233-2, a procaryotic expression vector was purchased from lPharmacia. PKK233-2 was used to transform 16 in a laq-q host, E. coli cell JM105 or RB791. The cells 17 were selected in I~~f9 minimal media containing lug/ml 18 thiamine, prior to using them for transformation. All 19 chemicals utilized in the synthesis of oligonucleotides were from Applied Biosystems Inc. T4 polynucleotide 21 kinase, DNA ligase, and Klenow fragment of E. coli DNA
22 polymerase I were obtained from New England Biolabs.
23 Restriction endonucleases, PMSF and IPTG were from 24 Boehringer Mannheim, Bethesda Research Laboratories and Promega respectively.
26 DNA SYNTHESIS. PLASMID CONSTRUCTION AND SCREENING:
27 DNA fragments were synthesized using a ABI DNA
28 synthesizer (model 381A). All synthetic fragments were 29 purified by electrophoresis in a 12~ polyacrylamide/8M
t urea sequencing gel. DrJA was visualized by W-shadowing 2 and full-length fragments were eluted from the gel as 3 known in the art. The full-length fragments were checked 4 for their purity using standard techniques.
Appropriate complementary fragments were mixed in 6 equimolar concentrations, annealed, kinased and ligated 7 as described elsewhere. The efficiency of ligation was 8 monitored by polyacrylamide gel lectrophoresis. The 9 linearized plasmid and the protease gene in appropriate concentrations were ligated and used for transformation 11 of E. coli, JM1~05. Recombinant clones were screened by 12 colony hybridization using a 62 by fragment labelled by 13 kinasing. Smal~1 scale isolation of plasmid DNA from the 14 recombinant clones was.performed by the boiling method and the size of the inserts was visualized by 16 autoradiography after labelling the 3' recessed terminal 17 using the Klenow fragment of E. coli DNA polymerase.
19 The polyclonal antibodies were raised in rabbits against (i) a complete synthetic sequence of 1 to 99 21 aminoacids of the HIV-1 protease and (ii) a 22 tridecapeptide corresponding to the C-terminus of the 23 protease.
E. coli cells bearing the appropriate plasmid 26 construct were grown to log phase, induced, and lysed by 27 sonication. Total cell extracts were analysed by 28 NaDodS04/PAGE and subjected to immunoblot analysis.
1 ASSAY FOR THE ACTIVITY OF THE EXPRESSED PROTEASE:
2 Oligopeptides were synthesized in a Peptide 3 Synthesizer (Applied Biosystems Model 430A), according to 4 the method prev3.ously published (Copeland and Oroszlan, S 1981). The cleavage products were analysed by RP-HPLC.on 6 a uBondapak Clg column (Waters Associates). Peak 7 fractions were analysed for amino-acid composition using 8 a Pico-Tag*amino acid analyser (Waters Associates).
9 E.'~AMPLE 1 This example represents the preferred embodiment.
11 RESULTS:
12 SYNTHESIS OF THE FULL-LENGTH PROTEASE GENE:
13 The nucleotide sequence of the protease gene was 14 taken from Ratner et al. The sequence in the pol open 1S reading frame for the protease gene starts at nucleotide 16 1609 and ends at: 1906, for coding 99 aminoacids. This 17 sequence and its complement were synthesized as five 18 individual fragments of approximately 60 bases as shown 19 in Figure 2. Th.e 3' averhangs of 4 bases (shown in lower case) were provided for the fragments to selectively 21 ligate the appropriate fragments to form the correct 22 coding sequence. Translational initiation codon ATG and 23 termination codon TAA were provided at the appropriate 24 ends of the protease gene. A sequence was added to provide a protrusion at the S' end of the gene, having a 26 cohesive end compatible to the restriction enzyme site 27 Ncol. The S' protrustion at the 3' end of the gene was 28 added to provide. a Hind3 compatible end. The 29 complementary strands (not shown) were provided with 3' overhangs to match the. coding strands.
* Trade Mark ca~F'i~: y i 1340'48 EXPRESSION OF THE SYNTHETIC HIV-1 PROTEASE GENE IN E. COLI
Three clones (PR-C, PR-H, and PR-J) bearing the correct coding sequence of 297bp in the expression vector PKK233-2 were analyzed for expression to select conditions for the optimal induction of the gene. 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 NaDodS04/PAGE transferred to nitrocellulose and subjected to immu:noblot 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. With increasing concentrations of inducer IPTG, 1-5 represents mM concentration of IPTG at 0.28, 0.56, 1.12, 2.24, and 4.48, respectively. Figure 3C showns the analysis after 60 minutes of induction with 1mM IPTG and lysing the cells in various buffers. B1 denotes lysis of cells in 50mM Tris-HC1 at pH 7.0, 150mM NaCl, 1mM EDTA, 1mM PMST, 1mM DTT and 0.5 percent NP-40. B2 is the same as Bl, but without N~aCl and EDTA. B3 is in 50 mM potassium phosphate at pH 6.0, 1mM PMSF and 1mM DTT. B4 is the same as B3 with a pH of 6.5. G denotes control cells bearing just the plasmid PKK233-2 and induced. Three times more protein was loaded in this lane. Positions of protein molecular weight 'markers are inducated on the left in kilodaltons.
12 This sequence was synthesized as five fragments~using 13 the DNA synthesizer. Complementary strands corresponding 14 to these five fragments were also synthesized. The 3' overhangs of four bases were provided for appropriate 16 sequences to efficiently ligate each of the five 17 fragments and to provide the correct coding sequence of 18 the protease gene. Nucleotide ATG were added to the 19 fragment corresponding to the 5' end of the gene and TAA
at the 3' end.
21 A procaryotac expression vector was used to clone and 22 then to express the synthetic sequence coding for the 23 protease. The expression can be in prokaryotes 24 (bacteria) or in other appropriate expression systems.
Recombinant clones screened by colony hybridization using 26 a labelled fragment (62bp) spanning the internal region 27 of the protease gene. Positive clones were further 28 analyzed for thE: size of the insert. Clones which 29 answered positi~ae were induced for expression and analyzed in Western blots to determine the protein 31 product using specific antibodies. Figure 1 gives an 32 example.
1 Of the clones screened so far, 3 clones have been 2 identified to express a product of ll.Skd, reacting 3 against specific antibodies as illustrated in Figure 1.
4 Conditions for the induction of a protease gene were S studied in E. coli and optimized. The inventors have 6 shown that the gene product has specific protease 7 activity, as it is capable of cleaving both synthetic and 8 natural substrates. The enzyme has been purified by 9 specific column chromatographic techniques, including affinity chromotography. The method of this invention 11 can produce enough active protease to study the structure 12 of the protease, its mechanisms of action, with a goal of 13 devising specific inhibitors to this enzyme, of a 14 therapeutic application for the treatment of the diseases, such as AIDS, caused by the viruses. Other 16 embodiments of this invention can utilize a gene to 17 express another protease such as the following gene for 18 the HIV-2 protease.
GTAGAAGTCTTGTTAGACACAGGGGCTGACGACTCAATAGTAGCAGGAATAGAGTTA
23 ACAGGCGACACCCAA'TCAACATTTTTGGCAGAAATATTCTGACAGCCTTAGGCATGT
Figure 1 demonstrates the expression of the HIV
26 protease in E. coli. Cells transformed with the 27 synthetic sequence of HIV protease in an appropriate 28 expression vector were induced and the bacterial lysate 29 was electrophor.esed in SDS-PAGE. After transfer of proteins into a nitrocellulose membrane, immunoblotting 31 procedure was performed using the specific antibody to 32 the HIV protease. Detection of Ag-Ab complex was made 33 using 1125 protein A. The autoradiograph lane A
_7_ 1340'~4~
1 represents E. coli transformed with the plasmid, and 2 lanes B and C coli transformed with the plasmid E.
3 bearing syntheticDNA encoding the HIV protease. On the 4 left are protein molecular weight markers in kilodalton.
S The 11.5 kd band is the protease.
6 The synthetic DNA of the invention also obviates any 7 need to manipulate (infectious) viral material and 8 overcomes limitations in the quantities which can be 9 obtained by other means.
EXAMPLES
11 The following materials and methods were used to 12 perform the examples.
13 PLASMID, BACTERIAL STRAINS, AND CHEMICALS:
14 Plasmid PKK233-2, a procaryotic expression vector was purchased from lPharmacia. PKK233-2 was used to transform 16 in a laq-q host, E. coli cell JM105 or RB791. The cells 17 were selected in I~~f9 minimal media containing lug/ml 18 thiamine, prior to using them for transformation. All 19 chemicals utilized in the synthesis of oligonucleotides were from Applied Biosystems Inc. T4 polynucleotide 21 kinase, DNA ligase, and Klenow fragment of E. coli DNA
22 polymerase I were obtained from New England Biolabs.
23 Restriction endonucleases, PMSF and IPTG were from 24 Boehringer Mannheim, Bethesda Research Laboratories and Promega respectively.
26 DNA SYNTHESIS. PLASMID CONSTRUCTION AND SCREENING:
27 DNA fragments were synthesized using a ABI DNA
28 synthesizer (model 381A). All synthetic fragments were 29 purified by electrophoresis in a 12~ polyacrylamide/8M
t urea sequencing gel. DrJA was visualized by W-shadowing 2 and full-length fragments were eluted from the gel as 3 known in the art. The full-length fragments were checked 4 for their purity using standard techniques.
Appropriate complementary fragments were mixed in 6 equimolar concentrations, annealed, kinased and ligated 7 as described elsewhere. The efficiency of ligation was 8 monitored by polyacrylamide gel lectrophoresis. The 9 linearized plasmid and the protease gene in appropriate concentrations were ligated and used for transformation 11 of E. coli, JM1~05. Recombinant clones were screened by 12 colony hybridization using a 62 by fragment labelled by 13 kinasing. Smal~1 scale isolation of plasmid DNA from the 14 recombinant clones was.performed by the boiling method and the size of the inserts was visualized by 16 autoradiography after labelling the 3' recessed terminal 17 using the Klenow fragment of E. coli DNA polymerase.
19 The polyclonal antibodies were raised in rabbits against (i) a complete synthetic sequence of 1 to 99 21 aminoacids of the HIV-1 protease and (ii) a 22 tridecapeptide corresponding to the C-terminus of the 23 protease.
E. coli cells bearing the appropriate plasmid 26 construct were grown to log phase, induced, and lysed by 27 sonication. Total cell extracts were analysed by 28 NaDodS04/PAGE and subjected to immunoblot analysis.
1 ASSAY FOR THE ACTIVITY OF THE EXPRESSED PROTEASE:
2 Oligopeptides were synthesized in a Peptide 3 Synthesizer (Applied Biosystems Model 430A), according to 4 the method prev3.ously published (Copeland and Oroszlan, S 1981). The cleavage products were analysed by RP-HPLC.on 6 a uBondapak Clg column (Waters Associates). Peak 7 fractions were analysed for amino-acid composition using 8 a Pico-Tag*amino acid analyser (Waters Associates).
9 E.'~AMPLE 1 This example represents the preferred embodiment.
11 RESULTS:
12 SYNTHESIS OF THE FULL-LENGTH PROTEASE GENE:
13 The nucleotide sequence of the protease gene was 14 taken from Ratner et al. The sequence in the pol open 1S reading frame for the protease gene starts at nucleotide 16 1609 and ends at: 1906, for coding 99 aminoacids. This 17 sequence and its complement were synthesized as five 18 individual fragments of approximately 60 bases as shown 19 in Figure 2. Th.e 3' averhangs of 4 bases (shown in lower case) were provided for the fragments to selectively 21 ligate the appropriate fragments to form the correct 22 coding sequence. Translational initiation codon ATG and 23 termination codon TAA were provided at the appropriate 24 ends of the protease gene. A sequence was added to provide a protrusion at the S' end of the gene, having a 26 cohesive end compatible to the restriction enzyme site 27 Ncol. The S' protrustion at the 3' end of the gene was 28 added to provide. a Hind3 compatible end. The 29 complementary strands (not shown) were provided with 3' overhangs to match the. coding strands.
* Trade Mark ca~F'i~: y i 1340'48 EXPRESSION OF THE SYNTHETIC HIV-1 PROTEASE GENE IN E. COLI
Three clones (PR-C, PR-H, and PR-J) bearing the correct coding sequence of 297bp in the expression vector PKK233-2 were analyzed for expression to select conditions for the optimal induction of the gene. 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 NaDodS04/PAGE transferred to nitrocellulose and subjected to immu:noblot 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. With increasing concentrations of inducer IPTG, 1-5 represents mM concentration of IPTG at 0.28, 0.56, 1.12, 2.24, and 4.48, respectively. Figure 3C showns the analysis after 60 minutes of induction with 1mM IPTG and lysing the cells in various buffers. B1 denotes lysis of cells in 50mM Tris-HC1 at pH 7.0, 150mM NaCl, 1mM EDTA, 1mM PMST, 1mM DTT and 0.5 percent NP-40. B2 is the same as Bl, but without N~aCl and EDTA. B3 is in 50 mM potassium phosphate at pH 6.0, 1mM PMSF and 1mM DTT. B4 is the same as B3 with a pH of 6.5. G denotes control cells bearing just the plasmid PKK233-2 and induced. Three times more protein was loaded in this lane. Positions of protein molecular weight 'markers are inducated on the left in kilodaltons.
E. coli cells bearing plasmid PP~-C were grown in 2 Luria broth to an optical density of 0.4 A600nm, and then 3 induced at various periods of time for expression from 4 the trc promotor by adding IPTG (isopropyl-beta-D-thiog-alactopyranoside) at a concentration of 0.4mM as seen in 6 Figure 3A. The cloned gene expressed a single, unfused 7 protein band of 11.5kd. Expression was maximal after 30 8 minutes of induction. This level decreased to about 25 9 percent at 60 minutes. There was no detectable expression after 120 minutes of induction and at 0 11 minutes. This pattern of induction was similar in the 12 other clones (PR-H and PR-J) that were analyzed (not 13 shown). _.
14 The results of the induction for 30 minutes with varying concentrations of inducer are shown in Figure 16 3B. Induction 'with IPTG in the range of 1mM to 4mM
17 resulted in maximum amount of expression. Similar data 18 were obtained on clones PR-H and PR-J (not shown).
19 In order to select the conditions that efficiently solubilize the protease for enzymatic analysis, different 21 buffer systems were used for the lysis of cells (clone 22 PR-C) after optimal induction with 1mM IPTG. It was 23 observed that sonication in a buffer system of 50mM
24 Tris-cl at pH 7..5, 1mM DTT, 1mM PMSF and 0.5% nonidet P-40 released 50 to 70 percent of the protease in the 26 soluble fraction (Figure 3C). This was estimated by 27 Western blot analysis aliquots of soluble extract and 28 insoluble pellet for the content of the expressed 29 product.
DEMONSTRATION OF SPECIFIC PROTEOLYTIC ACTIVITY
31 Figure 4 illustrates the activity of the expressed 32 protease using a synthetic peptide as a substrate.
Protease assays were carried out with 22.Sug of bacterial 2 lysate at 37oC obtained from clone PR-C, induced (A,B,C), 3 uninduced (D), and control cells bearing just he plasmid t 4 PKK233-2 (data not shown). THe nonapeptide was used as a S substrate in re action buffer (0.25 M potassium 6 phosphate), pH 7.0, 0.5 percent (v/v) NP 40, percent 7 (v/v) glycerol, S mM Dithiotreit and 2 M NaCl. Aliquots 8 of 25 ul each (B) 3 were taken at 0 hours (A), 1 hour 9 hours (C) and hours (D) analyzed by RP-HPLC. S denotes the substrate nd P1 and P2, cleavage products 1 and 2 a 11 respectively.
12 To assess the activity of the cloned HIV-1 Protease a 13 synthetic nonapeptide corresponding to the HIV-1 p17-p24 14 cleavage site (~Henderson, et al. 1988) was used as a substrate (4E). The substrate in reaction buffer was 16 mixed with aliquots of various cell extracts (see 17 description of Figure 4 above) and incubated at 37oC.
18 Equal eliquots of incubation mixture were taken at 19 various time points and analyzed by RP-HPLC. The substrate in the 0 hour sample eluted as a single peak as 21 shown in Figure 4A. After incubation for 1 hour, two 22 newly appearing peaks, products labelled P1 and P2, can 23 be seen, correlating with a signifant decrease of the 24 substrate peak. Subsequent amino acid analysis of the recovered peaks demonstrated that product 1 and product 2 26 corresponded to the expected cleavage products as shown 27 in Table 1 proving a Tyr-Pro bond cleavage, which is the 28 determined natural cleavage site. Extended incubation 29 for 3 hours showed a .further decrease of the substrate peak and substantial increase in the peak height of 31 product 1, indi<:ating progression of the hydrolysis of 32 the Tyr-Pro bond. However, the peak of product 1 seems 33 to be smaller as expected since the absorbance of the 34 tetrapeptide Pro-Ile-Val-Glu-NH2 is substantially smaller than that of the pentapeptide having a free 2 COOH-terminal t;yrosine. An increase of product 1 and 2 3 after 3 hours o:E incubation showed a corresponding 4 decrease of the substrate peak.
No cleavage products have been detected in reactions 6 using extracts :From uninduced cells, clone PR-C (Figure 7 4D) and of control cells (control plasmid PKK233-2; data 8 not shown). There was no decrease in the substrate peak 9 even after 6 hours of incubation (Figure 4D) indicating that the nonapeptide is resistent to degradation by 11 bacterial proteases. This makes this substrate 12 especially useful for assaying viral protease activities 13 in crude extracts, facilitating purification and 14 isolation of the protease.
The amino acid composition data for the substrate and 16 its cleavage products are shown in Table 1. The amounts 17 of observed amino acids correspond clearly to the 18 expected amounts demonstrating that the cleavage occurs 19 at the expected cleavage site of the synthetic peptide corresponding to the p17-p24 site of the gag precursor.
,..-.
o ~ -1 J ~1 w w ~c -s w N
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17 resulted in maximum amount of expression. Similar data 18 were obtained on clones PR-H and PR-J (not shown).
19 In order to select the conditions that efficiently solubilize the protease for enzymatic analysis, different 21 buffer systems were used for the lysis of cells (clone 22 PR-C) after optimal induction with 1mM IPTG. It was 23 observed that sonication in a buffer system of 50mM
24 Tris-cl at pH 7..5, 1mM DTT, 1mM PMSF and 0.5% nonidet P-40 released 50 to 70 percent of the protease in the 26 soluble fraction (Figure 3C). This was estimated by 27 Western blot analysis aliquots of soluble extract and 28 insoluble pellet for the content of the expressed 29 product.
DEMONSTRATION OF SPECIFIC PROTEOLYTIC ACTIVITY
31 Figure 4 illustrates the activity of the expressed 32 protease using a synthetic peptide as a substrate.
Protease assays were carried out with 22.Sug of bacterial 2 lysate at 37oC obtained from clone PR-C, induced (A,B,C), 3 uninduced (D), and control cells bearing just he plasmid t 4 PKK233-2 (data not shown). THe nonapeptide was used as a S substrate in re action buffer (0.25 M potassium 6 phosphate), pH 7.0, 0.5 percent (v/v) NP 40, percent 7 (v/v) glycerol, S mM Dithiotreit and 2 M NaCl. Aliquots 8 of 25 ul each (B) 3 were taken at 0 hours (A), 1 hour 9 hours (C) and hours (D) analyzed by RP-HPLC. S denotes the substrate nd P1 and P2, cleavage products 1 and 2 a 11 respectively.
12 To assess the activity of the cloned HIV-1 Protease a 13 synthetic nonapeptide corresponding to the HIV-1 p17-p24 14 cleavage site (~Henderson, et al. 1988) was used as a substrate (4E). The substrate in reaction buffer was 16 mixed with aliquots of various cell extracts (see 17 description of Figure 4 above) and incubated at 37oC.
18 Equal eliquots of incubation mixture were taken at 19 various time points and analyzed by RP-HPLC. The substrate in the 0 hour sample eluted as a single peak as 21 shown in Figure 4A. After incubation for 1 hour, two 22 newly appearing peaks, products labelled P1 and P2, can 23 be seen, correlating with a signifant decrease of the 24 substrate peak. Subsequent amino acid analysis of the recovered peaks demonstrated that product 1 and product 2 26 corresponded to the expected cleavage products as shown 27 in Table 1 proving a Tyr-Pro bond cleavage, which is the 28 determined natural cleavage site. Extended incubation 29 for 3 hours showed a .further decrease of the substrate peak and substantial increase in the peak height of 31 product 1, indi<:ating progression of the hydrolysis of 32 the Tyr-Pro bond. However, the peak of product 1 seems 33 to be smaller as expected since the absorbance of the 34 tetrapeptide Pro-Ile-Val-Glu-NH2 is substantially smaller than that of the pentapeptide having a free 2 COOH-terminal t;yrosine. An increase of product 1 and 2 3 after 3 hours o:E incubation showed a corresponding 4 decrease of the substrate peak.
No cleavage products have been detected in reactions 6 using extracts :From uninduced cells, clone PR-C (Figure 7 4D) and of control cells (control plasmid PKK233-2; data 8 not shown). There was no decrease in the substrate peak 9 even after 6 hours of incubation (Figure 4D) indicating that the nonapeptide is resistent to degradation by 11 bacterial proteases. This makes this substrate 12 especially useful for assaying viral protease activities 13 in crude extracts, facilitating purification and 14 isolation of the protease.
The amino acid composition data for the substrate and 16 its cleavage products are shown in Table 1. The amounts 17 of observed amino acids correspond clearly to the 18 expected amounts demonstrating that the cleavage occurs 19 at the expected cleavage site of the synthetic peptide corresponding to the p17-p24 site of the gag precursor.
,..-.
o ~ -1 J ~1 w w ~c -s w N
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w 7o w a o n -. 0 0 0 ~-. o .--. o -s o < o -0.~o~--ooo c~ a cn w .-. o ..r -s c o ..r -s * * ~ c~
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c a 'v c w r+ o .-.. .... o .-. r.
.... ...
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Claims (6)
- Claim 1. A method of producing a recombinant protein having proteolytic activity comprising the step of:
or allelic or species variation thereof, capable of encoding an amino acid sequence having the same amino acid sequence as the protein having the proteolytic activity, (b) culturing the resultant host cells of step a) under conditions such that the sequence or allelic or species variations thereof, is expressed and thereby producing the protein; and (c) isolating the protein. - Claim 2. The method of claim 1 wherein the protein is isolated by affinity chromatography.
- Claim 3. The method of claim 1 wherein the host cell is E
coli. - Claim 4. A construct comprising:
(a) a DNA gene sequence of or allelic or species variations thereof capable of encoding the same amino acid sequence as said DNA gene sequence, said sequence having proteolytic activity; and (b) an expression vector. - Claim 5. The construct of claim 4 further comprising a regulatory sequence operatively linked to the DNA gene sequence or allelic or species variation thereof.
- Claim 6. A gene for encoding a protease of human immunodeficiency virus comprising:
a substantially pure, double-stranded nucleotide sequence of which the coding sequence: is
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US21830488A | 1988-07-13 | 1988-07-13 | |
| US218,304 | 1988-07-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340748C true CA1340748C (en) | 1999-09-14 |
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ID=22814563
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000604978A Expired - Lifetime CA1340748C (en) | 1988-07-13 | 1989-07-06 | Synthetic hiv protease gene and method for its expression |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0425580A4 (en) |
| JP (1) | JPH04500602A (en) |
| AU (1) | AU635216B2 (en) |
| CA (1) | CA1340748C (en) |
| IL (1) | IL90947A (en) |
| WO (1) | WO1990000556A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2104576C (en) * | 1991-12-24 | 1997-04-15 | George Kannourakis | A method for the treatment of tumours and sarcomas |
| US6071895A (en) * | 1992-03-11 | 2000-06-06 | Narhex Limited | Polar-substituted hydrocarbons |
| ATE253050T1 (en) | 1992-03-11 | 2003-11-15 | Narhex Ltd | AMINE DERIVATIVES OF OXO- AND HYDROXY-SUBSTITUTED HYDROCARBONS |
| US5888992A (en) * | 1992-03-11 | 1999-03-30 | Narhex Limited | Polar substituted hydrocarbons |
| BR9306058A (en) * | 1992-03-11 | 1997-11-18 | Narhex Ltd | Amine derivatives of oxo- and hydroxy-substituted hydrocarbons |
| US6001558A (en) * | 1997-06-25 | 1999-12-14 | Ortho Clinical Diagnostics, Inc. | Amplification and detection of HIV-1 and/or HIV 2 |
| ATE384795T1 (en) | 1998-12-31 | 2008-02-15 | Novartis Vaccines & Diagnostic | MODIFIED HIV ENV POLYPEPTIDES |
| WO2000039304A2 (en) | 1998-12-31 | 2000-07-06 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
| EP1141313A2 (en) | 1998-12-31 | 2001-10-10 | Chiron Corporation | Improved expression of hiv polypeptides and production of virus-like particles |
| US7935805B1 (en) | 1998-12-31 | 2011-05-03 | Novartis Vaccines & Diagnostics, Inc | Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof |
| EP1427806A4 (en) | 2001-08-31 | 2006-04-26 | Chiron Corp | Polynucleotides encoding antigenic hiv type b polypeptides, polypeptides and uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
-
1989
- 1989-07-06 CA CA000604978A patent/CA1340748C/en not_active Expired - Lifetime
- 1989-07-12 IL IL90947A patent/IL90947A/en active IP Right Grant
- 1989-07-13 JP JP1508775A patent/JPH04500602A/en active Pending
- 1989-07-13 EP EP19890909316 patent/EP0425580A4/en active Pending
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| IL90947A (en) | 1997-06-10 |
| WO1990000556A1 (en) | 1990-01-25 |
| JPH04500602A (en) | 1992-02-06 |
| IL90947A0 (en) | 1990-02-09 |
| EP0425580A4 (en) | 1992-05-06 |
| AU635216B2 (en) | 1993-03-18 |
| AU4058589A (en) | 1990-02-05 |
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