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WO1999043792A1 - Proteine de recombinaison presentant une activite polymerase dependant de l'arn du virus de l'hepatite c humain et procede de production de cette derniere - Google Patents

Proteine de recombinaison presentant une activite polymerase dependant de l'arn du virus de l'hepatite c humain et procede de production de cette derniere Download PDF

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Publication number
WO1999043792A1
WO1999043792A1 PCT/JP1998/004204 JP9804204W WO9943792A1 WO 1999043792 A1 WO1999043792 A1 WO 1999043792A1 JP 9804204 W JP9804204 W JP 9804204W WO 9943792 A1 WO9943792 A1 WO 9943792A1
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Prior art keywords
ns5bt
rna
protein
activity
gst
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PCT/JP1998/004204
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English (en)
Japanese (ja)
Inventor
Seishi Murakami
Tatsuya Yamashita
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Eli Lilly And Company
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Publication of WO1999043792A1 publication Critical patent/WO1999043792A1/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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a soluble type I protein having an RNA-dependent RNA polymerase activity of hepatitis C virus.
  • Cffl- 1 Flame Virus is a + strand RNA virus isolated and identified in 1989 by the Chiron group as the causative virus of non-A, non-B hepatitis. Epidemiological studies estimate that the HCV-infected population worldwide is about 1%. Important routes of transmission other than those via blood, such as blood transfusion, are undecided, and it is estimated that the contribution of vertical transmission between parents and children is low. Prevention of infection by blood transfusion has achieved epoch-making results with the HCV immunodiagnosis developed by Chiron.
  • HCV is an RNA virus, it causes long-lasting infections in the liver and causes chronic hepatitis.
  • HCV chronic hepatitis for more than 30 years has been the leading cause of subsequent hepatocellular carcinoma (liver cancer), and in Japan at least 75% of ⁇ ff cancers develop on the background of HCV hepatitis. I have.
  • HCV-related liver cancer develops through a series of courses, so that the risk of onset can be predicted from the course of the disease state. Therefore, blocking the growth of HCV virus is expected to be effective in alleviating HCV hepatitis and preventing liver cancer development.
  • viral proteins are synthesized as a single long polypeptide (see Figure 1), which is cleaved by proteolytic enzymes encoded by the host and HCV genes during synthesis to produce viral structural and nonstructural proteins. .
  • proteolytic enzymes encoded by the host and HCV genes during synthesis to produce viral structural and nonstructural proteins.
  • studies on NS2 and NS3 aimed at inhibiting the protein cleavage process by the proteins encoded by HCV NS2 and NS3 have been carried out, In the situation where the development of the drug is progressing, 5
  • NS5 ⁇ is the central enzyme of HCV replication.
  • RNA-dependent RNA polymerase (hereinafter referred to as RdRP) (see Fig. 1)
  • RdRP RNA-dependent RNA polymerase
  • the present inventors have found that an anchor region exists in the C-terminal region of NS5B protein having the RNA-dependent RNA polymerase (hereinafter simply referred to as RdRP! Activity of human hepatitis C virus. After examining its role, the fusion of a soluble protein (NS5Bt), which deletes a certain number of amino acids from the C-terminal region and maintains its activity, with other polypeptides.
  • NS5Bt a soluble protein
  • the present inventors have found that expression of a protein in a host (for example, Escherichia coli) results in a soluble recombinant NS5Bt protein having RdRP activity, thereby completing the present invention.
  • NS5B becomes an insoluble fraction in the protein purification process due to the presence of the anchor region, whereas the NS5Bt mutant lacking the anchor region retains the R d RP activity while retaining the soluble fraction.
  • the presence of RdRP in the cells allowed the cells to be recovered and purified from the transformants without losing their RdRP activity (Example 2 of Fiber, see Fig. 10).
  • the present invention relates to a DNA encoding a polypeptide having polymerase activity derived from human hepatitis C virus RNA-dependent RNA polymerase, and a second polypeptide other than the polypeptide (eg, glutathione S
  • a host cell is transformed with the present vector containing DNA encoding transferase (hereinafter referred to as GST), the resulting transformant is cultured, and a fusion protein having polymerase activity is recovered from the medium.
  • GST DNA encoding transferase
  • a fusion protein having polymerase activity is recovered from the medium.
  • separating and recovering a polypeptide having polymerase activity from the fusion protein It is intended to provide a method for producing merase, and a soluble or non-fusion protein having a soluble recombinant RdRP activity produced in such a manner.
  • the method of the present invention has made it possible for the first time to provide a large amount of a purified recombinant recombinant NS5B protein having RdRP activity.
  • the soluble polypeptide derived from RdRP of human hepatitis C virus used in the present invention is generally a polypeptide in which the C-terminal region including all or a part of the anchor region of RdRP has been deleted.
  • NS5B of human hepatitis C virus has Rd RP activity in the HCV genome, the term "NS5B"
  • NS5Bt A soluble polypeptide (protein) derived from NS5B and derived from NS5B and having an RdRP activity with the C-terminal amino acid deleted is referred to as NS5Bt.
  • NS 5BJ and“ NS 5B t ” may represent a DNA coding for the NS 5 BS.TJiNS 5 B t protein.
  • FIG. 1 is a schematic diagram showing the structure of the HCV polyprotein precursor and its NS5B protein.
  • the upper part is a schematic diagram showing the structure of the HCV polyprotein precursor, and the middle part is the hydrophobic profile (Kyte and Doolittle) of the NS5B protein.
  • the anchor area estimated by the SOSU I program is shaded.
  • the lower panel is a schematic diagram showing the structures of expression constructs of various GST-NS5B fusion proteins.
  • the amino acid numbers in the figure correspond to the amino acid numbers on the HCV polyprotein precursor.
  • NS5B has all of the amino acids of NS5B
  • NS5Bt lacks the 2989-3010 21 amino acids
  • NS5Bt-ml has the GDD motif replaced with VDD
  • NS5Bt-m2 and NS5Bt-m3 have a cluster of salt S3 ⁇ 4 residues replaced by alanine
  • NS5B-m4 has a mutation in the putative anchor region.
  • FIG. 2 is a restriction map of the GST fusion protein expression plasmid, pGENKS.
  • Plastic Smid pGENKS encodes a consensus sequence for protein kinase A, a thrombin cleavage recognition site, and multicloning sites (EcoR I, Sacl, Kpn I, Xma I, Sall, BamH I), and a GST upstream of this. There is a DNA sequence to encode.
  • FIG. 3 is a schematic diagram of the construction of plasmid pGENKS.
  • the location of the GST-NS5Bt fusion protein cut by Tobin is underlined.
  • the nucleotide sequence of DNA shown in FIG. 3 and the amino acid sequence encoded thereby are shown in SEQ ID NO: 3.
  • Fig. 4 shows the steps of purification of GST-NS5Bt expressed in E. coli.A is separated from SDS obtained from culture by 10% PAGE and stained by Coomassie staining method (CBB). It is a mimetic diagram of the result of having performed.
  • CBB Coomassie staining method
  • Lane 1 is whole cell extract; Lane 2 is centrifuged supernatant of sonicated product; Lane 3 is cell-free extract after passing through DEAES-marked hacel; Lane 4 is eluate from daltathione sepharose 4B column Lane 5 shows the eluate from the poly (U) Sepharose column; Lane 6 shows the non-fused rNS5Bt after thrombin treatment.
  • B is a mimetic diagram of the results of Western blotting using an anti-NS5B antibody, and C is a western blotting result using an anti-GST antibody.
  • Fig. 5 shows the results of analyzing the purified GST-NS5Bt protein corresponding to the band at 95 Kda in lane 5 in Fig. 4A by Western blotting using the serum of a chronic hepatitis patient infected with HCV1b type b and the serum of a normal person
  • Lane 1 shows purified GST-NS5Bt
  • Lane 2 shows only the control GST protein.
  • the arrow points to the 95 kDa position.
  • FIG. 6 is a graph showing the RdRP activity of purified GST-NS5Bt in UMP uptake assays.
  • A uses poly (A) or poly (dA) as a template and oligo (U) 14 or oligo (dT) as a primer, and examines substrate specificity for [a- 32 P] UMP or O-P] dTTP It is a graph showing the result,
  • B shows the time course of the reaction at 25 and 37 °
  • C is a graph showing the relationship with the amount of GST-NS5Bt. .
  • Figure 7 shows UMP incorporation of purified GST-NS 5Bt under various reaction conditions.
  • 1 is a graph showing RdRP activity.
  • A shows the relationship between pH
  • B shows temperature
  • C shows the relationship between KC1 concentration and UMP uptake.
  • FIG. 8 is a graph showing Rd RP activity based on UMP incorporation of purified GST-NS5Bt under various reaction conditions.
  • Figure, D is shows a relationship between the Mg 2+ ion
  • E is Zn 2+ concentration and (0, 10, 25 and 5 0 // M) and UMP incorporation.
  • FIG. 9 is a diagram showing the results of an RNA synthesis assay using RNA transcribed in a test tube.
  • A is a schematic diagram of the construction of an RNA template
  • B is a reaction between the RNA transcribed in a test tube as a template and a primer at 30 ° C for 2 hours.
  • the RNA product is extracted with an organic solvent, and then precipitated with ethanol. It is a mimetic diagram of the electrophoresis pattern obtained by separation and purification by 8% PAGE containing 8M urea.
  • FIG. 10 is a micrograph showing the localization of NS5Bt expressed in mammalian cells.
  • A full length NS5B,
  • B GFP-NS5Bt,
  • C GFP-NS5B-m4,
  • D GFP alone, HLE transfected with an expression plasmid that encodes 3 shows the results of observing the localization of the germ product in Example 2 under a fluorescence microscope.
  • HCV-JK1 cDNA encodes 3010 polyprotein precursors, which are processed by viral and host proteases into at least 10 products .
  • NS5B which is located at C5 ⁇ ffi and is composed of 591 amino acids, is thought to encode RdRP polymerase by sequence comparison with other viruses (Honda, M., S. Kaneko, M. Unoura, K.
  • nucleotide sequence of the DNA encoding NS5B consisting of 591 amino acids and the amino acid sequence of O3 ⁇ 4t are shown in SEQ ID NOs: 1 and 2 in the sequence listing.
  • nucleotide sequence of DNA is shown in large letters, but they are shown in lowercase letters in the corresponding sequence numbers in the sequence listing below. Represents the same DNA as the nucleotide sequence shown.
  • recombinant NS5Bt having R d RP activity is produced using NS5B of HCV—JK1 cDNA as a starting material.
  • NS5B encoding RdRP derived from HCV
  • an active recombinant NS5Bt protein can be produced in the same manner as described herein. That is, if the amino acid sequence of the desired HCV-derived NS5B is known, the anchor region is identified from the sequence.If not, the sequence is determined as usual, and then the sequence is determined. The anchor region can be specified based on the sequence.
  • the number of amino acids to be deleted is appropriately selected depending on the HCV protein from which NS5B is derived.
  • the DNA encoding NS5Bt ie, the first polypeptide
  • the fusion protein with the appropriate second polypeptide is obtained.
  • An expression vector encoding is constructed, a host cell is transformed, and the transformant is cultured to recover an active product. Such methods are within the ordinary skill in the art.
  • the second polypeptide preferably has properties that are advantageous for purification and recovery of the fusion protein, and furthermore, if it is possible to separate NS5Bt from the fusion protein. More preferred.
  • the soluble polypeptide derived from the human hepatitis C virus Rd RP is an amino acid sequence represented by SEQ ID NOS: 1 and 2 in the sequence listing. Has a sequence from 1 to 570, or an amino acid sequence in which amino acid is deleted, substituted or added in the sequence, and has strong polymerase activity Things.
  • the recombinant R d RP of the present invention is novel and useful for the prevention and treatment of hepatitis C. Accordingly, the present invention provides a DNA encoding the recombinant RdRP, an expression vector containing the DNA, and a transformant transformed with the expression vector.
  • the NS5B protein derived from HCV-JKlc DNA and the NS5Bt protein derived therefrom are simply referred to as NS5B and NS5Bt.
  • the fusion protein of NS5Bt and glutathione S transferase (hereinafter referred to as GST) is called GST-NS5Bt, and the recombinant NS5Bt produced by the method of the present invention is called GST-NS5Bt.
  • the protein is called rNS5Bt or NS5Bt.
  • expression vector and the term “expression plasmid J” are used interchangeably.
  • the HCV-JK1 cDNA encodes 3010 polyprotein precursors and encodes a length of 0113 ⁇ 4?] ⁇ 358 is composed of the most C-terminal 591 amino acid (Fig. 1 ).
  • the present inventors have expressed a fusion protein of this amino acid-deficient mutant (NS5Bt) with GST and GST in a host cell by gene recombination, and extremely efficiently. It was possible to obtain a fused or unfused soluble NS5Bt protein having RdRP activity.
  • NS 5B consisting of 591 amino acids and a certain number of amino acids deleted from the C-terminal side
  • the obtained DNA encoding NS5Bt can be synthesized or obtained from known HCV-JK1c DNA by restriction enzyme digestion or PCR.
  • the number of amino acids to be deleted from NS5B described in SEQ ID NOS: 1 and 2 is the number of amino acids from the amino acid at position 591 at the C-terminal to the amino acid at position 571.
  • An example of such a polypeptide is a polypeptide with 21 amino acids deleted.
  • the object of the present invention is not only the C-terminal-deleted polypeptide derived from SEQ ID NOS: 1 and 2 (NS5Bt), but also a polypeptide having the desired RdRP activity. Mutants having R d RP activity in the amino acid sequence are also useful. Such a mutant can be induced, for example, by introducing a deletion, substitution, and Z or insertion of an amino acid into a polypeptide consisting of the amino acid sequence of amino acids 1 to 570 by a method known to those skilled in the art. . Deletions and additions include deletions and Zs or additions of amino acids at the N and Z or c termini. Screening of the obtained mutant can be performed by a known method or by the method described in Reference Example 2 described later.
  • the DNA encoding NS5Bt obtained by any method and the DNA encoding the second polypeptide, GST, are ligated with an appropriate expression vector to construct an expression vector for the fusion protein.
  • an expression vector is prepared by ligating the DNA of NS5Bt and DNA of GST in advance, and then inserting the DNA into an appropriate expression vector, or as a fusion protein, as described in Examples below.
  • GST and DNA encoding NS5Bt A suitable GST expression vector [eg, pG ENK1 (Murakami, S. et al., 1994, J. Biol. Chem. 269: 15118- Et al., 1997, Virology 231: 119-129] by inserting the DNA of NS5Bt.
  • the plasmid pGENKS described below is a protein kinase It encodes the consensus kinase kinase site of ZeA, the thrombin cleavage site and additional multiple cloning sites (EcoRI, Sad, Kpnl, Xmal, Sail and BamHI). GST is upstream of the multi-cloning site (MCS) of the GEN KS vector. Since the DNA to be inserted is inserted, it is suitable for the purpose of the present invention. However, the present invention can be practiced with any other expression plasmid.
  • a suitable host cell eg, Escherichia coli
  • a suitable host cell is transformed with the obtained expression vector and cultured in a suitable medium to produce a target fusion protein.
  • the cell-free extract was adsorbed to a Glutathion Sepharose 4B column (manufactured by Pharmacia), and phosphate buffered saline (PBS) containing 1% Triton X-100, followed by DTT After washing with Tris-hydrochloric acid buffer, daltathione was eluted with tf ⁇ buffer, and a large amount of high-purity GST-NS5Bt could be obtained.
  • NS 5 B t protein of the type can be separated.
  • Plasmids, various restriction enzymes, T4DN ligase, and other enzymes used in the following examples were obtained from commercial products and used according to the supplier's instructions.
  • DNA cloning, construction of each plasmid, transformation of a host, culture of the transformant, and recovery of the enzyme from the culture were carried out according to methods known to those skilled in the art or described in the literature.
  • Anti-hematosis was induced in the egret by subcutaneous injection of 200 # g of purified bacterial hexahistidine-labeled NS5Bt in egret in complete Freund's adjuvant (Sigma Chemicals Co. Ltd.) .
  • the IgG fraction of the antiserum was purified using a Protein A Sepharose column according to the instructions of the supplier (Pharmacia LB Co. Ltd.). Both antiserum and purified IgG fraction were used for Western blotting.
  • Sample prepared from E. coli or mammalian cell extract (lysate)
  • RNA-dependent RNA polymerase activity of samples containing the same were measured in principle by the following method. At the same time, other polymerase activities (reverse transcriptase activity, DNA-dependent RNA polymerase activity, DNA-dependent DNA polymerase activity) and terminal transfectase (TT) activity were also measured.
  • the standard reaction (10 / zl) is 20 mM Tris-HCl (pH 7.5), 5 nM MgCl 2 , ImM DTT, lmM EDTA, 20 U Rase inhibitor (Wako 2 ⁇ Ci [a- 32 P] UT P (800 Ci / nmol, Amersham Co. Ltd.), 10 ⁇ M UTP, 10 / xg / ml in a buffer containing poly (A) and oligo (U) 14. Purified NS 5 B t , And incubated for 2 hours at 25, then filter the sample with DE 81 filter (Whatman
  • the Rd RP activity was determined by measuring the total reaction volume of 40 ⁇ l [20 DAI Tris-HCl (pH 7.5), 5 ⁇ MgCl 2> lmM DTT, lm EDTA, 20U RNase inhibitor (Wako Fiber), 50 # g / ral actinomycin D (Sigma Co. Ltd.), 5 / zCi [a- 32 P] UT P and 0.5 n each of the remaining NT class (ie, ATP, CTP and GTP), and l O g / ral RNA This was performed using the template and. The concentration of restriction nucleotides was adjusted to ⁇ . After addition of the purified NS5Bt sample, the reaction mixture was incubated at 30 for 2 hours. After incubation, an equal volume of 2X proteinase K buffer (300 mM
  • RNA products were extracted with phenol-cloth form (1: 1), subjected to ethanol precipitation, and analyzed with 8 M urea- 8% PAGE. After electrophoresis, the gel is dried, exposed to an imaging plate, and the BAS 1000 Bioimage Analyzer
  • the GST fusion protein expression plasmid pGENKS was constructed using pGENKl (Murakami, S. et al., 1994, J. Biol. Chem. 269: 15118-15123; Yi, ⁇ ⁇ — ⁇ . Et al., 1997,
  • Virology 231: 119-129 was digested with EcoRI and BamHI to prepare pGENKl EcoRI / BamHI beta, and synthetic oligonucleotides encoding the following multicloning sites were inserted.
  • the above synthetic oligonucleotide was annealed with 65 in the presence of NaC1, digested with EcoR I-BamHI, extracted with phenol monoclonal form, ethanoled, and dissolved in Tris EDTA (hereinafter TE). Ligation Ligation was conducted at ligation high (Toyobo) according to the supplier's instructions. Thereafter, Escherichia coli strain XLl-Blue was transformed by an ordinary method to prepare plasmid pGENKS.
  • Blasmid p GENKS encodes a consensus sequence for protein kinase A, a thrombin cleavage ⁇ site, and multiple cloning sites (EcoR I, Sac I, Kpn I, Xma I, Sall, BamHI). There is a DNA sequence encoding GST.
  • Figure 2 shows a restriction map of this plasmid pGENKS, and
  • Figure 3 shows a schematic diagram of its construction.
  • Transformation of Escherichia coli is performed by a usual molecular biological method. Add the plasmid DNA or ligation reaction mixture to 60 / il of competent E. coli, incubate on ice for 30 minutes, then incubate at 42 for about 1 minute, then on ice for 2 minutes, add SOC solution, After incubating at 373 ⁇ 4 for 45 minutes, plate on an LB plate containing 100 gZml ampicillin for approximately 12 to 20 hours. Culture in C to obtain a transformant.
  • the NS5B-containing HCV JK1 subgenomic cDNA (Honda, M. et al., 1 " 3 , Arch Virol. 128: 163-169) was converted to a protein synthesis initiation codon, artificial Sac I and Sal I restriction sites. Subcloning was performed by PCR using the following set of primers having
  • PCR was performed according to the following procedure according to the supplier's instructions. That is, a series of processing consisting of (943 ⁇ 4, 1 minute; 58, 1 minute; 723 ⁇ 4, 2 minutes) was performed for 30 cycles. By this operation, about 1.7 kb of NS5B cDNA was amplified by PCR. Then, this PCR product was subjected to low-melting point agarose gel electrophoresis, a DNA band was cut out, and recovered using ordinary phenol, black form, and ethanol precipitation. The recovered cDNA was treated with Sac I and Sal I restriction enzymes (Takara Shuzo), treated with phenol, black mouth form, precipitated with ethanol, purified, and used for the next step.
  • Sac I and Sal I restriction enzymes (Takara Shuzo)
  • a plasmid expressing the full length NS5B was first constructed.
  • pGENKS is treated with Sacl and SalI restriction enzymes to create pGENKS SacI / SalI vector, and the NS5BcDNASacIZSalI fragment obtained in (1) is ligated as a T4 DNA ligase kit.
  • 'Hi Toyobo
  • a ligation reaction was performed according to the supplier's instructions, and the cells were transformed into Escherichia coli to obtain pGENKSZNS 5B.
  • NS5Bt in which the C-terminal 21 amino acids have been deleted awakens the pG ENKS NS5B obtained in (2) above, with NS5BFor and the next primer (NS5BtRev):
  • the Escherichia coli strain was transformed by the method described in 2 above, cultured by the following method, and the expression product was purified.
  • Escherichia coli strain obtained by transformation with pGENKS / NS 5Bt plasmid
  • This culture 11 was centrifuged, E. coli was harvested, and washed once with a phosphorous-digested diet: fek (PBS). Then, the pellet was mixed with InM dithiothreitol (DTT) and
  • Noffer A 32ral PBS containing 1% Triton X-100
  • the suspension was sonicated on ice until viscous and centrifuged at 2,000 g for 20 minutes.
  • the supernatant 1 (S1) and the pellet were separated, and the supernatant (S1) was left on ice.
  • the pellet was suspended in 32 ml of buffer A containing 1.0 M NaCl, the suspension was similarly sonicated, and the centrifuged supernatant was collected (S 2), mixed with the above SI, and mixed with buffer A.
  • the concentration of NaCl was adjusted to 0.33M with, and supernatant 3 (S3) was obtained.
  • the fraction obtained in 3) above was mixed with 1 ml of glutathione Sepharose 4B beads (Pharmacia Biotech Co. Ltd.) equilibrated in buffer A, and the protein was incubated at 43 ⁇ 4 for 1 hour. The beads were absorbed. The beads were then buffer A, then ImM DTT containing 5 OmM Tris-HCl (pH
  • the NaCl concentration of the eluate was adjusted to 15 OmM, and then applied to a heparin sepharose column (Pharmacia Biotech Co. Ltd,) equilibrated with an elution buffer containing 15 OmM NaCl. After washing with LG buffer containing 15 OmM NaCl, the column was washed with 0 buffer with NaCl concentration of 10001 ⁇ 1 ⁇ 11 ⁇ [20raM
  • Tris-HCl pH 7.5
  • lmM EDTA 5 mM DTT
  • 20% glycerol 0.5% Triton X-100
  • the eluate was analyzed by 10% SDS-PAGE. As a result, the eluate was eluted in a wide range of NaCl concentration of GST-NS5B3500 mM to 90 On.
  • GST-NS5Bt was eluted in the range of 1 ⁇ & ⁇ 1 concentration 500111 ⁇ 1 to 70 OmM. This fraction was collected and dialyzed against an LG buffer containing 150 mM NaCl (FIG. 4A, lane 5).
  • NS5Bt was treated with thrombin and cut with an artificial consensus sequence at the GST binding site (Figure 3 underlined). That is, daltathione beads (Daltathione Sepharose 4B beads) to which GST-NS5Bt was bound were converted into thrombin cleavage buffer ((50 mM Tris-HCl (pH 7.5), 150 nAf NaCl, 2.5 mM CaCl 2 , 1% After washing thoroughly with Triton X-100), then thrombin (50 U) (Pharmacia)
  • thrombin cleavage buffer containing thrombin cleavage buffer was treated overnight at 4 with GST carrier to allow fusion of GST with NS5Bt protein. Centrifuge the beads and remove the supernatant containing NS5Bt protein for 15 minutes. The sample was dialyzed against LG buffer containing OmM NaCl, and the obtained sample was analyzed by measuring protein concentration and Western blotting (FIG. 4A, lane 6). In addition, polymerase activity was also measured.
  • FIG. 4 shows changes in the degree of purification in each purification step, and Table 1 shows changes in the polymerase activity.
  • A is a simulated view of the result of separating the sample by SDS-10% PAGE and staining with Coomassie staining method (CBB).
  • Lane 1 is whole cell extract
  • Lane 2 is the centrifuged supernatant of the sonicated product
  • Lane 3 is the cell-free extract after passing through DEAE Sephacel
  • Lane 4 is the eluate from the G / letathion Sepharose 4 B column
  • 5 is an eluate from a poly (U) Sepharose column
  • lane 6 represents non-fused rNS5Bt after thrombin treatment.
  • B is a mimetic diagram of the results obtained by subjecting the samples from these six purification stages to Western blotting using an anti-NS5B antibody, and C being subjected to Western blotting using an anti-GST antibody.
  • lane 5 a 95 kDa recombinant GST-NS5Bt protein was obtained in 90% or more.
  • Lane 6 of Figure 4A shows the approximately 63 kDa protein of the non-bound fraction of the thrombin digest. A double band is observed at the position corresponding to the protein. This band is specifically recognized and recognized by anti-NS5BtIgG in Western blotting, but is not recognized by anti-GST IgG, indicating that it is a non-fused rNS5Bt protein. Was confirmed (Figs. 4B and C).
  • Table 1 shows that UMP uptake of purified GST-NS5Bt was detected, indicating that the relative activity was at least 10,000-fold or more.
  • the non-fused NS5Bt truncated tombin was slightly lower than the fusion protein, but showed UMP uptake activity.
  • the final purified fusion protein (GST-NS5Bt) and non-fusion protein (NS5Bt) were dialyzed extensively against LG-buffer containing 150 mM NaC1. Saved in.
  • the polymerase activity of the purified GST-NS5Bt having a purity of 90% or more purified in the above (6) was analyzed according to the method described in Reference Example 2. That is, as a substrate - an [ ⁇ 32 P] UMP or [tx- 32 P] dTMP incorporation was determined using purified GST- NS 5B t (90 ng) .
  • Figure 6 shows the results.
  • A indicates the substrate specificity when poly (A) or poly (dA) was used as a template and oligo (U) 14 or oligo (dT) was used as a primer.
  • B is the time course of the reaction at 25 and 37.
  • C shows the relationship between the amount of GST-NS5Bt and UMP uptake.
  • NS5Bt is not harmful and its concentration is increased to 200 ⁇ g ml and 500 ⁇ g / ml, respectively. It was not inhibited by addition (Table 2).
  • Controls contain equal amounts of ethanol.
  • UMP uptake is inhibited by KC1 concentrations above 100 mM KC1 and is strictly dependent on Mg2 + ions, with optimal concentrations of Mg2 + ranging from 2.5 to 5 ⁇ (Figure 8D). ).
  • CHAPS concentrations are 0.1 lmM and lmM_3 ⁇ 4t 10 mM, respectively.
  • RNA synthesis assay using HCV RNA as template and primer In the UMP uptake test described in (7) above, a synthetic template and primer were used, but GST-NS5Bt used HCV RNA as a template and primer. We examined whether it was possible or not. The reaction was carried out at 30 ° C. for 2 hours using the same solution as the above RNA polymerase assay.
  • UTR of HCV which should function as a replication initiation site, was divided into three regions, as shown in Figure 9A.
  • 5B Bg is the region from the Bglll site of NS 5B to 3, UTR; poly (U) is a poly (U) stretch; and 3, X is a highly conserved sequence at the 3, terminus of HCV genomic RNA. Yes, contains 3 'X which has been suggested to play an important role in viral replication
  • RNA templates were prepared by in vitro transcription. These plasmids are It was constructed by PCR using the following synthetic oligonucleotide primers.
  • pGEM3zf (+) / NS5BBg is the primer:
  • pGEM3zf (+) / poly (U) is an oligonucleotide:
  • pGEM3zf (+) / 3'X is a set of oligonucleotides:
  • pGEM3zf (+) / 3'X was digested with BamHI or EcoRI and linearized.
  • pNKF LAG constructed by the method described in Test Example 1 described below was digested with Bglll and used to prepare control RNA.
  • RNA concentration was measured by J2 spectrophotometry, adjusted to 1 / igZj "1 with RNase-free distilled water, and stored at 1-20.
  • RNA samples The quality of the RNA samples was confirmed by electrophoresis using MOPS denaturing gel or urea denaturing polyacrylamide gel.
  • RNA-dependent RNA polymerase (RdRP) was assayed.
  • the RNA product was treated with proteinase K, extracted with phenol monochloroform, precipitated with ethanol, and finally separated and purified by 8% * 8% PAGE.
  • the size of each RNA is as shown in Fig. 9A.
  • FIG. 9B The results are shown in FIG. 9B. A new, radioactive-labeled RNA larger than the input RNA was detected (Fig. 9B, lanes 1, 3, and 4).
  • RNA synthesis by GST-NS5Bt of the present invention required all four types of liponucleotides as substrates, and no uptake was detected when UTP or CTP ⁇ M was used as a substrate.
  • HCV RNA and control RNA acted as templates and primers (FIG. 9).
  • RNA synthesis ability using 3'X of HCV3 and UTR is slightly lower than other RNAs (Fig. 9, lanes 1 and 4; Compared to 3), 11 ⁇ 3 ⁇ 3,1; the 0 ⁇ (1)) stretch of the cho 1 ⁇ did not act as a template or primer by itself (Fig. 9, lane 2).
  • the recombinant GST-NS5Bt and the recombinant NS5Bt expressed by the Escherichia coli transformant of the present invention share a common RdRP. It can be seen that the protein is an active soluble protein having properties (requirements of template and primer, Mg 2+ dependence, and optimal reaction conditions, etc.).
  • the GST-NS5Bt and NS5Bt of the present invention also differ from the recombinant NS5B expressed in insect cells in that they do not exhibit terminal transferase activity. .
  • a substitution mutant in the GDD motif of HCV NS5B (NS5Bt-ml), a mutant in YRHRAR (NS5Bt-m2) and a mutant in CGYRRCR (NS5Bt-m3) were designed (Fig. 1). See).
  • RHRAR was substituted with AAAAAA (500-505 (2919-2924)), and CGYRR CR was replaced with AAAAAA (274-280 (2693-2699)).
  • DNAs encoding these substitution mutants were prepared by PCR mutagenesis with overlapping extensions using primers designed for mutagenesis, NS5BFor and O ⁇ SSBtRev (Yi, M.-K. Et al., 1997, Virology.
  • a substitution mutant in the terminal region of NS5B was obtained by PCR using NS5BFor and NS5Bm4Rev having the following sequence as primers.
  • GCGGATCCTC ACCGGTTGGGG AGCAGGTAGAT
  • NS5B hepatocellular carcinoma cell line HLE cells, HepG2 cells, non-liver cells.
  • mammalian cells hepatocellular carcinoma cell line HLE cells, HepG2 cells, non-liver cells.
  • COS 1 cells Transient expression system using cancer cells
  • an expression plasmid encoding a fusion protein of green fluorescent protein (GFP) and full-length NS5B, NS5Bt and NS5B-m4 was constructed.
  • HLE of mammalian cells was transformed with these plasmids and a plasmid encoding GFP warrior, and the localization of the expressed protein in the cells was examined.
  • GFP green fluorescent protein
  • GFP green fluorescent protein
  • AAGATATCGC GGCCGCATGGT GAGCAAGGGCG AG (GFPNotFor) (SEQ ID NO: 16) AAGGATCCGA ATTCTTGTACA GCTCGTCCAT (GFPEcoRev) (SEQ ID NO: 17) These have synthetic EcoRV, Notl and EcoRI sites, respectively.
  • the DNA fragment having the synthesized EcoRV and OamHI sites was treated with EcoRV and BamHI, and the EcoRI site of PSG5UTPL was blunt-ended with the Klenow fragment, and then inserted into the BamHI-digested vector to obtain pGFP.
  • pGFP vector to construct another mammalian expression vector, pNKF LAG.
  • a sequence encoding a FLAG-tag (label) epitope sequence having a region related to translation initiation was obtained from pF LAGHis / p53 (obtained from R. Roeder) using the PCR method described below. This DNA fragment is combined with the following set of primers:
  • Plasmid pNKF LAG contains EcoRI and BamHI sites as described above. And was constructed by replacing the p53 insert with a multiple cloning site.
  • NS5B DNA was inserted into the EcoRI and BamHI sites of these plasmids pNKFLAG and pGFP according to a conventional method, and the GFP-NS5B expression plasmid (pGFP / NS5B) or FLAG-labeled NS5B Expression plasmid
  • Immunostaining is performed by counterstaining with Evans Blue using standard methods to absorb absorbed egret anti-mouse IgG, biotinylated goat anti-pea IgG, and streptavidin-FITC (Amersham). went.
  • the result was a BX-50 fluorescence microscope equipped with a NI BARX WI B filter.
  • FIG. 10 shows that GFP-NS5B protein was mainly distributed around the cytoplasmic nuclear membrane and dispersed in the cytoplasm, but GFP alone diffused and localized in both the cytoplasm and nucleus.
  • Figure 10A In addition, GFP-NS5Bt protein mainly accumulates in the nucleus, and its localization was significantly affected by the C-terminal and truncated regions.
  • FIG. 10 the fluorescent intranuclear signal of GFP-NS5Bt is observed as a cluster of several clusters with large, spherical, lj-like shapes.
  • GFP-NS5B-m4 which has two substitution mutations designed to impair the function of the C-terminal anchor region of NS5B, is also localized in the nucleus and partially found in the cytoplasm.
  • Figure 10C Similar results were seen with other mammalian cells, COS1 cells or HepG2 cells. The above results indicate that the 21 amino acids at the C-terminus of NS5B play an important role in the subcellular localization of NS5B protein, and the role of anchors on the membrane is important. It was considered to indicate.

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Abstract

Un procédé de production d'une polymérase d'ARN dépendant de l'ARN de recombinaison consiste à transformer des cellules hôtes à l'aide d'un vecteur d'expression contenant un ADN issu d'une polymérase d'ARN dépendant de l'ARN d'un virus d'hépatite C humain et codant un polypeptide soluble ayant une activité polymérase et un autre ADN codant un deuxième polypeptide différent du polypeptide précédemment mentionné, à cultiver le transformant résultant et à prélever du milieu une protéine fusionnée présentant l'activité polymérase, et facultativement à effectuer ensuite la séparation d'avec la protéine fusionnée et la récupération du polypeptide présentant l'activité polymérase, puis à récupérer la polymérase d'ARN dépendant de l'ARN de recombinaison ainsi obtenu.
PCT/JP1998/004204 1998-02-27 1998-09-18 Proteine de recombinaison presentant une activite polymerase dependant de l'arn du virus de l'hepatite c humain et procede de production de cette derniere WO1999043792A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1995028243A1 (fr) * 1994-04-19 1995-10-26 United Technologies Corporation Refroidissement d'une aube de turbine a gaz
JPH09299079A (ja) * 1996-05-15 1997-11-25 Japan Energy Corp 組換え単純ヘルペスウイルス１型プロテアーゼおよびその製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028243A1 (fr) * 1994-04-19 1995-10-26 United Technologies Corporation Refroidissement d'une aube de turbine a gaz
JPH09299079A (ja) * 1996-05-15 1997-11-25 Japan Energy Corp 組換え単純ヘルペスウイルス１型プロテアーゼおよびその製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VOLKER LOHMANN et al., "Biochemical Properties of Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase and Identification of Amino Acid Sequence Motifs Essential for Enzymatic Activity", JOURNAL OF VIROLOGY, (1997), Vol. 71, No. 11, p. 8416-8428. *

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