WO1998012308A1 - Polypeptides solubles a activite de serine protease ns3 du virus de l'hepatite c et leur procede de preparation et d'isolement - Google Patents

Polypeptides solubles a activite de serine protease ns3 du virus de l'hepatite c et leur procede de preparation et d'isolement

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Publication number
WO1998012308A1
WO1998012308A1 PCT/IT1997/000228 IT9700228W WO9812308A1 WO 1998012308 A1 WO1998012308 A1 WO 1998012308A1 IT 9700228 W IT9700228 W IT 9700228W WO 9812308 A1 WO9812308 A1 WO 9812308A1
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WIPO (PCT)
Prior art keywords
hcv
protease
polypeptides
protein
pro
Prior art date
Application number
PCT/IT1997/000228
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English (en)
Inventor
Raffaele De Francesco
Anna Tramontano
Licia Tomei
Maria Chiara Nardi
Christian STEINKÜHLER
Andrea Urbani
Rosa Letizia Vitale
Stefano Colloca
Original Assignee
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A
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.)
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Application filed by Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A filed Critical Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A
Priority to JP10514467A priority Critical patent/JP2001500735A/ja
Priority to AU43970/97A priority patent/AU4397097A/en
Priority to EP97942190A priority patent/EP0950094A1/fr
Priority to CA002264487A priority patent/CA2264487A1/fr
Publication of WO1998012308A1 publication Critical patent/WO1998012308A1/fr

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    • 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

Definitions

  • HCV hepatitis C virus
  • NANB non-A, non-B hepatitis
  • HCV is an enveloped virus containing an RNA positive genome of approximately 9.4 kb. This virus is a member of the Flavivirida.e family, the other members of which are the pestiviruses and flaviviruses .
  • RNA genome of HCV has recently been sequenced. Comparison of sequences from the HCV genomes isolated in various parts of the world has shown that these sequences can be extremely heterogeneous. Most of the HCV genome is occupied by an open reading frame (ORF) that can vary between 9030 and 9099 nucleotides. This ORF codes for a single viral polyprotein, the length of which can obviously vary from 3010 to 3033 amino acids. During the virus infection cycle, the polyprotein is proteolytically processed into the individual gene products necessary for replication of the virus. The genes coding for HCV structural protein are located at the 5' end of the ORF, whereas the region coding for the non-structural proteins occupies the rest . i . of the ORF.
  • ORF open reading frame
  • the structural proteins consist of: C (core, 21 kDa) , El (envelope, gp37) and E2 (NS1, gp61) .
  • C is a non-glycosilate protein of 21 kDa, which probably forms the viral nucleocapsid.
  • the protein El is a glycoprotein of approximately 37 kDa and is believed to be a structural protein of the outer viral envelope.
  • E2 another membrane glycoprotein of 61 kDa, is probably a second structural protein of the outer envelope of the virus .
  • the non-structural region starts with NS2 (p24) , a hydrophobic protein of 24 kDa whose function is not known.
  • NS3 a protein of 68 kDa which follows NS2 in the polyprotein, has two functional domains: a serine protease domain in the first 180 amino-terminal araino acids and an RNA-dependent ATPase domain in the carboxy- terminal part .
  • the gene region corresponding to NS4 codes for NS4A (p6) , a membrane protein of 54 amino acids, and NS4B (p26) .
  • the gene corresponding to NS5 codes for two proteins, NS5A (p56) and NS5B (p65) , of 56 and 65 kDa, respectively. Recently it has been shown that the NS5B region has an RNA dependent RNA-poly erase activity (1) .
  • a first protease activity of HCV is responsible for the cleavage between NS2 and NS3. This activity is contained in a region comprising both a part of NS2 and the part of NS3 containing the serine protease domain, but does not use the same catalytic mechanism (3) .
  • the serine protease contained in the 180 amino acids at the amino-terminal of NS3 is responsible for cleavage at the junctions between NS3 and NS4A, between NS4A and NS4B, between NS4B and NS5A, and between NS5A and NS5B
  • N R methods and X-ray crystallography requires large amounts of soluble protein, and at the present time it is not possible to meet this request.
  • the simplest and most economical manner of obtaining large amounts of the desired polypeptide is expression of the corresponding gene in bacteria, and although there is a widespread availability of numerous eucaryotic promoters and methods for maximising the expression of heterologous genes in E. Coli, nevertheless an efficient production of the polypeptide in question, although necessary, might not be sufficient.
  • Many recombinant proteins do not fold the polypeptidic chain correctly when they are expressed in E . Coli. The result is the synthesis of polypeptides which are either degraded in the host cell, or are accumulated in an insoluble form in the so called inclusion bodies (15) .
  • proteins of viral origin or proteins that are toxic for the bacterial cell (as is the case for certain proteases of viral origin) there are insurmountable difficulties in producing them in a native, soluble form.
  • this method is based on the unexpected discovery that the NS3 serine protease domain, in its native conformation, binds a Zn ion. Because, as mentioned above, the structure of the HCV NS3 protease is not yet known, a structural model of the protein was prepared, to be used as a guide during experiments. However, the similarity of the NS3 protease to other serine proteases of known structure is extremely low (less than 15%) , which does not allow good alignment between sequences and as a result does not allow construction of a three-dimensional model based solely on homology.
  • the HCV NS3 protease actually has a metal content equivalent to one mole of zinc to each mole of protein, and as is the case in other proteins the zinc is necessary to enable the protein to take on its native structure and become catalytically active (20, 21) .
  • the NS3 protease has a binding site for a metal ion and that this binding site is so well preserved, even in viruses that are not phylogenetically close, opens the way to the study of antiviral therapeutic agents whose target site is this very region of the protein.
  • another viral protein that binds Zn 2+ ions that is to say the HIV virus nucleocapsid
  • An object of the present invention is therefore to provide a method for high-yield expression, in a native form, that is to say as a protein containing a bivalent metallic ion, and in a highly soluble form of the HCV N ⁇ 3 protease using heterologous expression systems, such as E. coli cells transformed using suitable genetic constructs and cultivated in a medium enriched with salts containing divalent metal ions.
  • a further object of the present invention is to provide a general method allowing preparation and isolation in a native, pure and highly soluble form, of large amounts of polypeptides containing Zn + , Co or
  • an additional object of the present invention is to provide a method that allows preparation and isolation in a native, pure and highly soluble form of large amounts of polypeptides with the protease activity of HCV NS3 , which are at the same time marked using stable heavy isotopes such as 13 c or 15 N , as required for experiments to determine the three- dimensional structure of the protein using NMR.
  • the present invention provides new genetic constructs for the expression, in E. coli cells, of modified polypeptides with the protease activity of HCV
  • a procedure for obtaining production of the NS3 serine protease domain in its native form, that is to say containing a bivalent metal ion, which is necessary for the structural integrity of the protein.
  • the innovation in the procedure consists in the addition to the culture medium in which the transformed bacterial cells are grown of compounds containing metals such as Zn, Co, Cd, Mn, Cu, Ni, Ag, Fe, Cr, Hg, Au, Pt , V. These compounds provide the culture medium with the ions required by the protein to take on its native structure.
  • the protein is found in its native, soluble form in the cytoplasm of the bacterial cells, instead of being held in the included bodies, from which it can only be obtained by applying difficult resolubilisation procedures .
  • a procedure is provided that makes it possible to replace the zinc ion in the protease, which is spectroscopically silent, with other ions (for example Co or Cd + ) , which are spectroscopically active, so as to permit the study of possible inhibitors capable of co-ordinating the metal contained in the protein and therefore of disturbing the bond between the protein and the metal .
  • bivalent metal ions to a minimum culture medium, containing glucose and ammonium salts enriched with 13C or 15N as the sole sources of carbon and nitrogen, respectively, makes it possible to obtain large amounts of soluble protein marked with stable heavy isotopes such as 13C or 15N.
  • This type of isotope enrichment is necessary to determine the structure using NMR techniques .
  • polypeptide sequences are provided that contain the NS3 serine protease domain of hepatitis C virus, suitably modified.
  • polypeptides are characterised in that they have at their C-terminal end a sequence of extremely hydrophilic amino acids, such as for example a series of lysines, which are not present in the original sequence.
  • a sequence of extremely hydrophilic amino acids such as for example a series of lysines
  • Subjects of the present invention are therefore: a) Isolated and purified polypeptides containing the HCV NS3 serine protease domain, characterised in that they have at their C-terminal end a tail of at least three lysines. b) A process for the preparation of polypeptides containing the HCV NS3 serine protease domain in a soluble form, of use for enzymological experiments, determination of the three-dimensional structure of the enzyme both by means of NMR and using X-ray crystallography, comprising the following operations:
  • a process for the renaturation in vitro of the above polypeptides characterised in that it comprises the following operations:
  • Figure 1 shows the alignment between the HCV NS3 serine protease sequence and the viruses GBV-A, GBV-B and GBV-C/HGV (Hcv, Hga, Hgb, Hgc) , with the poliovirus (Pol) 2A cysteine protease.
  • Amino acids conserved in the HCV proteases and in the viruses GBV-A, GBV-B and GBV-C/HGV are shaded.
  • the catalytic residues are underlined and the residues that bind zinc are indicated using the symbol _.
  • Figure 2 shows a diagrammatic model of the NS3 serine protease domain. In particular it shows the position within the structure of the amino acids involved in binding zinc (dark grey) and the catalytic triad
  • Figure 3 shows the effect of the zinc ion on HCV NS3 serine protease activity.
  • Figure 4 shows the effects of the zinc ion on the production of HCV NS3 protease as a soluble protein in E. coli on a minimum culture medium.
  • Column 2 refers to the results of the experiment carried out on the cells without inducing protease production (-IPTG) .
  • Columns 3, 4 and 5 indicate that in the absence of ZnCl 2 and following the induction of protease production (+IPTG) the protein remains locked in the insoluble portion (indicated by the abbreviation PT) .
  • the protease is found entirely in the soluble portion (indicated by the abbreviation SN) .
  • FIG. 5 shows the electronic spectrums of the HCV
  • Figure 5a shows the visible and near-UV spectrum of the Co 2+ -protease .
  • Figure 5b shows the UV absorption spectrums of the Zn + -protease and of the Cd * - protease.
  • the plasmids pT7-7(Pro BK-asK4), pT7-7(Pro H-asK4), pT7-7(Pro J-asK4) and pT7-7(Pro J8-asK4) were constructed to allow expression in E. coli of polypeptides characterised in that they have a sequence chosen from the ones in the group from SEQ ID NO : 1 to SEQ ID NO : 4.
  • the polypeptides contain the NS3 protease domain of various HCV isolates (BK, H, J and J8 , respectively) with the addition of a "tail" of four lysines at the C- terminal end.
  • pT7-7 (Pro BK-asK4) contains the sequence for HCV-BK (EMBL data bank access number: M58335) between the nucleotides 3411 and 3950, cloned in the vector pT7-7.
  • pT7-7 (Pro H-asK4) contains the sequence for HCV-H (EMBL data bank access number: M67463) between the nucleotides 3420 and 3959, cloned in the vector pT7-7.
  • pT7-7 (Pro J-asK4) contains the sequence for HCV-J (EMBL data bank access number: D90208) between the nucleotides 3408 and 3947, cloned in the vector pT7-7.
  • pT7-7 contains the sequence for HCV-J8 (EMBL data bank access number: D10988/D01221) between the nucleotides 3432 and 3971, cloned in the vector pT7- 7.
  • the expression vector pT7-7 is a derivative of pBR322 which contains, in addition to the gene for ⁇ lactamase and the replication origin of ColEl, the promotor and the ribosome binding site of the T7 bacteriophage 010 gene (24) .
  • the fragments coding for the HCV NS3 protease were cloned downstream of the T7 bacteriophage 010 promoter, in reading frame with the first ATG condon of the gene 10 protein of phage T7 using methods known to the art .
  • the cDNA fragment containing the sequence HCV-BK between nucleotides 3411 and 3950 was amplified by Polymerase Chain Reaction (PCR) , using the oligonucleotides PR0T(BK-K4)S (SEQ ID NO: 5) and PROT (BK- K4)AS (SEQ ID NO: 6) as primers.
  • the cDNA fragment so obtained was digested with the restriction enzyme Ndel , and cloned in pT7-7, which was first linearised with the restriction enzymes Ndel and S al .
  • the cDNA fragment containing the sequence HCV-H between nucleotides 3420 and 3959 was amplified by PCR, using the oligonucleotides PROT(H-K4)S (SEQ ID NO: 7) and PROT(H-K4)AS (SEQ ID NO: 8) as primers.
  • the cDNA fragment so obtained was digested with the restriction enzymes Ndel and EcoRI , and cloned in pT7-7, which was first linearised with the same restriction enzymes.
  • the cDNA fragment containing the sequence HCV-J between nucleotides 3408 and 3947 was amplified by PCR, using the oligonucleotides PROT(J-K4)S (SEQ ID NO: 9) and PROT(J-K4)AS (SEQ ID NO: 10) as primers.
  • the cDNA fragment so obtained was digested with the restriction enzymes Ndel and EcoRI , and cloned in pT7-7, which was first linearised with the same restriction enzymes.
  • the cDNA fragment containing the sequence HCV-J8 between nucleotides 3432 and 3971 was amplified by PCR, using the oligonucleotides PROT(J8-K4)S (SEQ ID NO:ll) and PROT(J8-K4)AS (SEQ ID NO: 12) as primers.
  • the cDNA fragment so obtained was digested with the restriction enzymes Ndel and EcoRI, and cloned in pT7-7, which was first linearised with the same restriction enzymes.
  • the plasmids pT7-7(Pro BK-asK4), ⁇ T7-7(Pro K-asK4) , pT7-7(Pro J-asK4) and pT7-7 (Pro J8-asK4) containing NS3 sequences also contain the gene for ⁇ -lactamase, which can be used as a selection marker for E. coli cells transformed with these plasmids.
  • the fragments were cloned downstream of the T7 bacteriophage promotor, in reading frame with the first ATG codon of the gene 10 protein of phage T7 using methods known to the art.
  • the plasmids pT7-7(Pro BK- asK4) , pT7-7(Pro H-asK4), pT7-7 (Pro J-asK4) and pT7-7(Pro J8-asK4) containing NS3 sequences also contain the gene for ⁇ -lactamase, which can be used as a selection marker for E. coli cells transformed with these plasmids.
  • the plasmids are then transformed in the E. coli strain BL21 (DE3), normally used for high levels of expression of genes cloned in expression vectors containing the T7 promotor.
  • the T7 polymerase gene is carried into the bacteriophage ⁇ DE3 , which is integrated into the chromosome of BL21 cells (25) .
  • Expression of the gene is induced by incubating the cultures at an A600 nm of 0.7-0.9 with 0.4 mM of isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG) for 3 hours at 20°C in LB culture medium additioned with ZnCl2 at a concentration that can vary from 50 ⁇ M to 1 mM.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the cells are harvested and washed in a saline phosphate buffer solution (20 mM sodium phosphate pH 7.5, 140 mM NaCl) , after which they are re-suspended in 25 mM sodium phosphate at pH 7.5, 10% glycerol, 500 mM NaCl , 10 mM DTT, 0.5% CHAPS (10 ml per 1 litre of culture medium) .
  • the cells are then lysated by passing twice through a "French pressure cell" and the homogenate obtained in this way is centrifugated at 100,000xg for 1 hour, while the nucleic acids are removed by precipitation with 0.5% polyethylenimine .
  • the supernatants are loaded onto a HiLoad 16/10 SP Sepharose High Performance column (Pharmacia) , and balanced with 50 mM of sodium phosphate at pH 7.5 , 5% glycerol, 3 m DTT, 0.1% CHAPS (buffer A).
  • the column had been washed repeatedly with buffer A and the protease was eluted by applying a gradient of from 0 to 0.6 M NaCl.
  • the fractions containing the protease were then collected and concentrated using a chamber for ultrafiltration under magnetic stirring, equipped with a YM-10 membrane (Amicon) .
  • the sample was then loaded onto an HR 26/60 HiLoad Superdex 75 column (Pharmacia) , balanced with buffer A, operating at a flow rate of 1 ml/min.
  • the fractions containing NS3 were collected and further purified on an HR 5/5 Mono S column (Pharmacia) , balanced with buffer B and operating at a flow rate of 1 ml/min.
  • the protease was eluted from the column in pure form applying a linear gradient of 0-0.6 NaCl in buffer A.
  • the concentration of the protein was estimated by determination of absorbancy at 280 nm using a coefficient of extinction deriving from the sequence data or from quantitative amino acid analysis. Both methods come to the same results, with an error factor of 10%.
  • the purity of the enzyme was ascertained on SDS polyacrylamide gel and by HPLC using an inverse phase
  • Vydac C4 column (4.6x250 mm, 5 mm, 300 A) .
  • the eluents used were H2O/0.1% TFA (A) and acetonitryl/0.1% TFA (B) .
  • a s nth tic pe tide ⁇ 1J amino aciu ⁇ was used as a substrate.
  • This peptide was derived from the cleavage sequence of the NS4A-NS4B junction (DEEMECSSHLPYK) .
  • a peptide with 14 amino acids corresponding to the central hydrophobic region of the protein NS4A (from position 21 to position 34) was used as a protease cofactor.
  • the peptides were synthesised by solid phase synthesis based on Fmoc chemistry. After washing and deprotection, the "raw" peptides were purified by HPLC to 98% purity. The identity of the peptides was determined by mass spectrometry .
  • the peptide solutions stored were prepared in DMSO and preserved at -80°C, furthermore the concentrations were determined by quantitative amino acid analysis carried out on samples hydrolysed with HC1.
  • the cleavage tests were carried out using 300 nM - 1.6 ⁇ M of enzyme in 30 1 of 50 mM Tris pH 7.5, 50% glycerol, 2% CHAPS, 30 mM DTT and appropriate amounts of substrate and/or peptide-NS4A at 22°C.
  • the reaction was stopped by addition of 70 ⁇ l of H20 containing 0.1% TFA.
  • Cleavage of the peptide substrate was determined by HPLC using a Merck-Hitachi chromatograph. After this, 90 ⁇ l of each sample were injected into an inverse phase Lichrospher C18 cartridge column (4x125 mm, 5 ⁇ m, Merck) and the fragments were separated using an acetonitryl gradient of 3-100% at 2%/min.
  • Tables 1 and 2 give the data for solubility and yield relating to the NS3 protease corresponding to various HCV virus isolated.
  • Table 1 gives the data for production of the various forms of protease both with and without the addition of four lysines at the C- terminal end, and both with and without the addition of ZnC12 in the culture medium. The data are expressed as the percentage of protein recovered in the soluble fraction of the cell extracts and the protein found in the included bodies.
  • Table 2 gives the yields and solubility of the various forms of protease, purified from E.
  • the modified proteases (BK-ASK4, J-ASK4, H-ASK4) are between 10 and 20 times more soluble and, when expressed in a culture medium containing an excess of ZnCl2, they give a yield up to 10 times greater than the respective proteases without the lysine tail .
  • the protease (at a concentration of 4 mg/ml) was dialysed for a period of at least 16 hours against a buffer containing 50 mM Tris/Hcl pH 7.5, 3 mM DTT, 10% glycerol, 0.1% CHAPS.
  • a Chelex-100 resin (2.5 g/1) was held in suspension in the dialysis buffer to prevent contamination by casual metal ions.
  • the protein was then hydrolysed with nitric acid and then used to determine the metal content .
  • the standardised Zn + , Co and Cd + solutions were purchased from Merck.
  • NS3 serine protease activity its proteolytic activity was first measured on a synthetic substrate peptide.
  • NS3 protease were denaturated by addition of TFA to a final concentration of 1%.
  • the denaturated protein was then purified on a Resouce RPC 3 ml column using an acetonitryl gradient of from 0% to 85% in the presence of
  • the apoprotein was diluted to a final concentration of 60 nM in the activity buffer containing the concentrations of ZnCl 2 shown in the graph and 10 mM DTT to prevent oxidation of the thiole groups. After an incubation period of 1 hour at 22°C the reaction was started by adding the substrate eptide at a concentration of 40 mM. The reaction was then made to proceed for another hour before taking the measurements. As shown in figure 3, reconstitution of the enzymatic activity depends on the concentration of zinc ions in the buffer. Maximum reactivation was observed at a ZnCl2 concentration of 25 ⁇ M.
  • HCV NS3 protease contains a structural zinc atom has been used to increase the production of soluble protein in bacterial cells (E. coli) and therefore to produce a protein in a form that can be used for experiments aimed at determining the structure by means of NMR.
  • determination of structure by means of NMR involves metabolic marking with 15 N and 13 c , to be carried out on a minimum culture medium, for example modified M9 culture medium (NH 4 ) 2 S0 4 lg/1, K-phosphate 100 mM, MgS0 4 0.5 mM, CaCl 2 0.5 ml ⁇ , biotin S ⁇ M, thiarnine 7 ⁇ M, a picillin 5 ⁇ g/ml, glucose 4 g/1, FeS0 4 .7H 2 0 13 ⁇ M) .
  • modified M9 culture medium NH 4 ) 2 S0 4 lg/1, K-phosphate 100 mM, MgS0 4 0.5 mM, CaCl 2 0.5 ml ⁇ , biotin S ⁇ M, thiarnine 7 ⁇ M, a picillin 5 ⁇ g/ml, glucose 4 g/1, FeS0 4 .7H 2 0 13 ⁇ M
  • FIG. 4 shows how the protease (at approximately 21 kDa - indicated in the figure by an arrow) is produced as an insoluble aggregate (PT) when the bacterial cells are grown in minimum culture medium without zinc (columns 3, 4 and 5) .
  • the Zn 2+ binding site of the HCV NS3 protease and zinc can be studied by replacing the zinc with metals that make spectroscopic studies possible.
  • the close binding of the structural zinc to the enzyme makes it difficult to remove the metal and replace it in vi tro .
  • the Zn * was replaced by Co 2+ and Cd 2+ by incorporation in vivo.
  • the bacterial cells (E. coli) were transformed with an appropriate expression vector and grown in minimum culture medium containing 100 M potassium phosphate at pH 7.0, 0.5 mM MgS0 4 , 0.5 mM CaCl 2 , 13 ⁇ M FeS0 4 , 7 ⁇ M thiamine, 6 ⁇ M biotin.
  • Glucose (4 g/1) and (NH4)2S04 (1 g/1) were used as sources of carbon and nitrogen, respectively.
  • the phosphate buffer was made to pass through a Chelex-100 column.
  • 50 mM of CoCl2 and CdCl2 were added, respectively, 20 minutes before addition of IPTG.
  • Purification of the Co + and Cd ⁇ -proteases was obtained using the procedure described in example 1, except for the fact that all the buffers used were treated with Chelex-100 resin (2.5 g/1) and the DTT was eliminated.
  • the protease containing Co + and Cd + was subjected to electronic absorption spectroscopic analysis .
  • the protease containing Co + shows a typical absorption spectrum in the visible region (figure 6a) , which indicates a binding site with a tetrahedral geometry
  • the two main bands at 640 nm and at 685 nm and the minimums at 585 nm and at 740 nm indicate d-d transitions.
  • the energy in these transitions and the molar extinction coefficients are characteristic of complexes with a distorted tetrahedral co-ordination geometry (27) .
  • the d-d transition energy is consistent, with a mixed sulphur-nitrogen co-ordination bond.
  • the centroide in the band corresponding to the d-d transition indicates a Co complex with a S3N bond (26) .
  • Lam P . Y . Jadhav P . K . , Eyermann C.J., Hodge C . , Ru Y., Bacheler L.T., Meek J.L., Otto M.J., Rayner M.M., Wong Y.N., Chang C.-H., Weber P.C, Jackson D.A., Sharpe T.R., Erickson-Viitanen S. (1994) Science 263:380-384. 15. Georgiou G. and Valax P. (1996) Current Opinion in Biotechnology 7:190-197.
  • NAME Pro BK-asK4
  • D OTHER INFORMATION: sequence for the NS3 protease of HCV-isolated BK.
  • Cys lie lie Thr Ser Leu Thr Gly Arg Asp Lys Asn Gin Val Glu Gly
  • Cys lie lie Thr Ser Leu Thr Gly Arg Asp Lys Asn Gin Val Glu Gly 20 25 30
  • Glu Val Gin lie Val Ser Thr Ala Thr Gin Thr Phe Leu Ala Thr Cys
  • NAME PROT(BK-K4)S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
  • NAME PROT (BK-K4 )
  • AS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTACTTCTTC TTCTTGCTAG CCCGCATAGT AGT 33
  • NAME PROT(H-K4)AS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TTTGAATTCC TACTTCTTCT TCTTGCTAGC TCTCATGGTT GT 42
  • NAME PROT(J-K4)S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTTCATATGG CGCCTATCAC GGCCTAT 27
  • NAME PROT(J-K4)AS (xi) SEQUENCE DESCRIPTION SEQ ID NO: 10:
  • MOLECULE TYPE Synthetic DNA
  • ANTISENSE No
  • NAME PROT(J8-K4)S (xi) SEQUENCE DESCRIPTION SEQ ID NO: 12: TTTGAATTCC TACTTCTTCT TCTTGCTAGC CCGTGTGGCG AC 42

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Abstract

La présente invention concerne la sérine protéase NS3 du virus de l'hépatite C et notamment l'observation que le domaine de la sérine protéase NS3, dans sa conformation native, lie un ion Zn2+ et que des ions métalliques bivalents sont nécessaires à l'intégrité structurelle de la protéine et à l'activité de l'enzyme. La présente invention concerne également des polypeptides recombinés comprenant des séquences de la protéase SN3 et caractérisés par une queue d'au moins trois lysines à leur extrémité C-terminale afin d'en augmenter la solubilité. Un autre sujet de l'invention àtrait à un nouveau procédé permettant l'expression desdits polypeptides, sous la forme de métalloprotéines, avec l'activité protéolyptique de la protéase SN3 du virus de l'hépatite C en une forme soluble et en une quantité suffisante pour permettre de rechercher à identifier des inhibiteurs et déterminer la structure tridimensionnelle de la protéase SN3. La figure 4 illustre les effets de l'ion zinc sur la production de la protéase SN3 du virus de l'hépatite C en tant que protéine soluble dans E.Coli dans un milieu de culture minimum.
PCT/IT1997/000228 1996-09-17 1997-09-17 Polypeptides solubles a activite de serine protease ns3 du virus de l'hepatite c et leur procede de preparation et d'isolement WO1998012308A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP10514467A JP2001500735A (ja) 1996-09-17 1997-09-17 Ｃ型肝炎ウィルスのｎｓ３セリンプロテアーゼの活性を有する可溶性ポリペプチド、およびそれらの製造および単離法
AU43970/97A AU4397097A (en) 1996-09-17 1997-09-17 Soluble polypeptides with activity of the ns3 serine protease of hepatitis c virus, and process for their preparation and isolation
EP97942190A EP0950094A1 (fr) 1996-09-17 1997-09-17 Polypeptides solubles a activite de serine protease ns3 du virus de l'hepatite c et leur procede de preparation et d'isolement
CA002264487A CA2264487A1 (fr) 1996-09-17 1997-09-17 Polypeptides solubles a activite de serine protease ns3 du virus de l'hepatite c et leur procede de preparation et d'isolement

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ITRM960632 IT1285158B1 (it) 1996-09-17 1996-09-17 Polipeptidi solubili con l'attivita' di serino-proteasi di ns3 del virus dell'epatite c, e procedimento per la loro preparazione e il

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2160046A1 (es) * 1998-03-30 2001-10-16 Hoffmann La Roche Derivados pentapeptidicos.
US6333186B1 (en) 1999-01-08 2001-12-25 Bristol-Myers Squibb Company Modified forms of Hepatitis C NS3 protease for facilitating inhibitor screening and structural studies of protease: inhibitor complexes
US7012066B2 (en) 2000-07-21 2006-03-14 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7169760B2 (en) 2000-07-21 2007-01-30 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7244721B2 (en) 2000-07-21 2007-07-17 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7705138B2 (en) * 2005-11-11 2010-04-27 Vertex Pharmaceuticals Incorporated Hepatitis C virus variants
US7884199B2 (en) 2003-10-27 2011-02-08 Vertex Pharmaceuticals Incorporated HCV NS3-NS4 protease resistance mutants
US8759026B2 (en) 2009-07-31 2014-06-24 Baxter International Inc. Methods for increasing recovery of an ADAMTS activity from a cell culture supernatant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992012992A2 (fr) * 1991-01-14 1992-08-06 James N. Gamble Institute Of Medical Research Polypeptides immunogeniques structuraux de base ayant des epitopes pour vhc, anticorps, sequences de polynucleotides, vaccins et methodes
WO1996036702A2 (fr) * 1995-05-12 1996-11-21 Schering Corporation Protease active soluble du virus de l'hepatite c
WO1997008304A2 (fr) * 1995-08-22 1997-03-06 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Methode de production, de purification et d'analyse de polypeptides par l'activite proteolytique de la vhc ns3 protease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992012992A2 (fr) * 1991-01-14 1992-08-06 James N. Gamble Institute Of Medical Research Polypeptides immunogeniques structuraux de base ayant des epitopes pour vhc, anticorps, sequences de polynucleotides, vaccins et methodes
WO1996036702A2 (fr) * 1995-05-12 1996-11-21 Schering Corporation Protease active soluble du virus de l'hepatite c
WO1997008304A2 (fr) * 1995-08-22 1997-03-06 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Methode de production, de purification et d'analyse de polypeptides par l'activite proteolytique de la vhc ns3 protease

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KHUDYAKOV YU.E. ET AL: "Linear B-Cell Epitopes of the NS3-NS4-NS% Proteins of the Hepatitis C Virus as modeled with Synthetic Peptides", VIROLOGY, vol. 206, 1995, pages 666 - 72, XP002003036 *
KIM D.W. ET AL.: "C-terminal Domain of Hepatitis C Virus NS3 protein contains an RNA helicas activity", BIOCHE.BIOPHYS.RESERARCH COMMUNICATIONS, vol. 215, no. 1, 1995, pages 160 - 166, XP002035618 *
TOMEI L. ET AL.: "NS3 is a Serine Protease required for processing of Hepatitis C Virus Polyprotein", J. VIROLOGY, vol. 67, no. 7, 1993, pages 4017 - 4026, XP000561255 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2160046A1 (es) * 1998-03-30 2001-10-16 Hoffmann La Roche Derivados pentapeptidicos.
US6333186B1 (en) 1999-01-08 2001-12-25 Bristol-Myers Squibb Company Modified forms of Hepatitis C NS3 protease for facilitating inhibitor screening and structural studies of protease: inhibitor complexes
US6800456B2 (en) 1999-01-08 2004-10-05 Bristol-Myers Squibb Company Modified forms of hepatitis C NS3 protease for facilitating inhibitor screening and structural studies of protease:inhibitor complexes
US7012066B2 (en) 2000-07-21 2006-03-14 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7169760B2 (en) 2000-07-21 2007-01-30 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7244721B2 (en) 2000-07-21 2007-07-17 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7595299B2 (en) 2000-07-21 2009-09-29 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
USRE43298E1 (en) 2000-07-21 2012-04-03 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
US7884199B2 (en) 2003-10-27 2011-02-08 Vertex Pharmaceuticals Incorporated HCV NS3-NS4 protease resistance mutants
US7705138B2 (en) * 2005-11-11 2010-04-27 Vertex Pharmaceuticals Incorporated Hepatitis C virus variants
US8501450B2 (en) 2005-11-11 2013-08-06 Vertex Pharmaceuticals Incorporated Hepatitis C virus variants
US8759026B2 (en) 2009-07-31 2014-06-24 Baxter International Inc. Methods for increasing recovery of an ADAMTS activity from a cell culture supernatant
US9127265B2 (en) 2009-07-31 2015-09-08 Baxalta Incorporated Cell culture medium for ADAMTS protein expression
US9441216B2 (en) 2009-07-31 2016-09-13 Baxalta Incorporated Cell culture medium for ADAMTS protein expression
US10072254B2 (en) 2009-07-31 2018-09-11 Baxalta Incorporated Cell culture methods for expressing ADAMTS13 protein
US10724024B2 (en) 2009-07-31 2020-07-28 Baxalta Incorporated Cell culture methods for expressing ADAMTS protein
US11254921B2 (en) 2009-07-31 2022-02-22 Takeda Pharmaceutical Company Limited ADAMTS13 protein cell culture supernatant

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Publication number Publication date
AU4397097A (en) 1998-04-14
EP0950094A1 (fr) 1999-10-20
ITRM960632A1 (it) 1998-03-17
JP2001500735A (ja) 2001-01-23
CA2264487A1 (fr) 1998-03-26
IT1285158B1 (it) 1998-06-03

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