| PATENT ASSIGNEE'S COUNTRY | Korea |
| UPDATE | 10.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 19.10.99 |
| PATENT TITLE |
HBV polymerase processes for preparation and uses for screening antiviral agents thereof |
| PATENT ABSTRACT |
The present invention relates to hepatitis B virus (hereinafter it refers to HBV) polymerase containing a histidine tag, RNase H enzyme derived from HBV polymerase and processes for preparation thereof. More particularly, the present invention relates to recombinant HBV polymerase, its RNase H domain with enzyme activity, expression vectors producing the enzymes in E. coli and processes for preparing the HBV polymerase and the RNase H enzyme which can be easily purified due to their histidine tags. And the present invention relates to uses of the HBV polymerase and the RNase H enzyme for screening antiviral agents. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 15.08.97 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Muller et al., "Co-expression of the Subunits of the Heterodimer of HIV-1 Reverse Transcriptase in Escherichia coli", J. Bio. Chem 264, No. 24, pp. 13975-13976 (Aug. 1989). Tan et al., "Inhibition of the RNase H Activity of HIV Reverse Transcriptase by Azidothymidylate", Biochemistry 30, No. 20, pp. 4831-4835 (May 1991). Smith, et al., "Purification and Characterization of an Active Human Immunodeficiency Virus Type 1 RNase H Domain", Journal of Virology 67, No. 7, pp. 4037-4049 (Jul. 1993). |
| PATENT CLAIMS |
What is claimed is: 1. A hepatitis B virus (HBV) polymerase containing a histidine tag. 2. The HBV polymerase according to claim 1, which is fused with maltose binding protein. 3. The HBV polymerase according to claim 2, which contains a histidine tag at the C-terminus. 4. A expression vector which produces the HBV polymerase of claim 1 in Escherichia coli. 5. The expression vector according to claim 4, which is the expression vector pMPH. 6. A E. coli transformant which is prepared by transforming host cells with the expression vector of claim 4. 7. The E. coli transformant according to claim 6, which is prepared by transforming host cells with the expression vector pMPH (KCCM-10084). 8. A process for preparing a hepatitis B virus (HBV) polymerase containing a histidine tag comprising steps as follows: (a) culturing the E. coli transformants of claim 6, (b) inducing the expression of the HBV polymerase, (c) performing histidine tag affinity column chromatography with crude extract of the E. coli transformant. 9. The process for preparing the HBV polymerase according to claim 8, comprising further steps as follows: (a) performing amylose affinity column chromatography, (b) treating with protease factor Xa. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION The present invention relates to hepatitis B virus (hereinafter it refers to HBV) polymerase containing a histidine tag, RNase H enzyme derived from HBV polymerase and processes for preparation thereof. More particularly, the present invention relates to recombinant HBV polymerase, its RNase H domain with enzyme activity, expression vectors producing the enzymes in E. coli and processes for preparing the HBV polymerase and the RNase H enzyme which can be easily purified due to their histidine tags. And the present invention relates to uses of the HBV polymerase and the RNase H enzyme for screening antiviral agents. HBV is the main virus among hepatitis viruses, which infects more than 300 million people worldwide. HBV causes acute or chronic hepatitis, which results in liver cirrhosis or liver cancer (Tiollais and Buenda, Scientific American, 264: 48-54, 1991; Blumberg, B. S., Background and perspective in advances in hepatitis research, F. V. Chisari, ed., New York, Mason publishing, 1984). Because of molecular characteristics of HBV and its close relation with liver diseases, various researches about HBV have been accomplished. HBV is a DNA virus, a member of the hepadnaviridae family, which has a spherical structure composed of nucleocapsid and core. HBV genome is a partially double stranded DNA of only 3.2 kb size, which is not a circular form. In detail, HBV genome is composed of four overlapped genes that are the polymerase (P) gene, the surface protein (HBsAg; S, pre-S1, pre-S2) gene, the core protein (HBcAg; pre-C, C) gene and X protein (HBx) gene. Among these genes, X protein gene encodes regulatory protein, and the other genes encode structural proteins of HBV. The polymerase gene occupies 80% of the total genome and encodes 94 KD-sized protein composed of 845 amino acids. HBV infects hepatic cells by the process described below. The specific receptor of the hepatic cell recognizes the surface protein on the surface of the virion particle and binds with them so as to draw the virion into the hepatic cell. Then HBV polymerase synthesizes the single-stranded part of partially double-stranded DNA in order to obtain complete HBV genome. And the HBV genome of 3.2 kb size is transcribed with cellular RNA polymerase to produce pre-genomic mRNA of about 3.5 kb, core protein (C) mRNA, surface protein mRNA and X protein mRNA. Viral proteins are translated from these mRNAs. Specially HBV polymerase synthesizes an RNA intermediate with its reverse transcriptase activity so as to provide a template for the DNA genome and make a replicasome structure with the pre-genomic mRNA, the core protein and the like, which is called encapsidation process. The HBV genome can be encapsidated easily since 3'-terminus of the polymerase containing continuous glutamic acid residues has affinity with nucleic acids. The above RNA intermediate in the replicasome serves as a template for minus strand DNA synthesis and then the full-length minus strand serves as a template for plus strand DNA synthesis by DNA-dependent DNA polymerase (DDDP) activity of the polymerase so as to make total pre-genomic mRNAs finally. By repeating the above process, more than 200-300 copies of the genomic DNA is maintained in pool and the viral proteins mentioned above are expressed (Tiollais and Buenda, Scientific American, 264: 48-54, 1991 ; Ganem, D. and Varmus, H. E., Annu. Rev. Biochem., 56: 651-693, 1987). Interestingly, HBV replicates its genome by using the RNA intermediate and reverse transcription even though it is a DNA virus. It is known that retrovirus exploits the reverse transcription to replicate its genome. Particularly, the polymerase of retrovirus is reported to be a multifunctional enzyme which shows DNA-dependent DNA polymerase activity, reverse transcriptase activity, and RNase H activity. It is remarkable that HBV polymerase contains a series of functions necessary for the replication of virus genome. Namely, the following functions: (i) protein primer, (ii) RNA-dependent DNA polymerase (RT), (iii) DNA-dependent DNA polymerase (DDDP), (iv) RNase H activity consist in one polypeptide. The reverse transcriptase activity of HBV polymerase was first reported by Kaplan et al., and has been exploited to elucidate the mecahnism of HBV replication. As mentioned above, a reverse transcriptase has an active RNase H domain commonly which recognizes RNA/DNA complex and hydrolyzes only the RNA strand selectively. The RNase H activity is indispensable to the reverse transcription, since the reverse transcriptase can replicate DNA continuously only after RNA intermediate is hydrolyzed by the RNase H activity. Although RNase H enzyme is known as a domain of the reverse transcriptase recently, RNase H enzyme was first discovered in the calf thymus by Hausen and Stein, and has been reported from various prokaryotes and eukaryotes (Stein, Hans and Hausen, P., Science, 166: 393-395, 1969). Generally speaking, an active RNase H domain of HBV polymerase is localized within its C-terminus. The amino acid sequence and nucleotide sequence of the polymerase were reported to be very similar to those of the polymerase of Moloney murine Leukemia Virus. In addition, an active RNase H domain of HBV polymerase was known to synthesize a plus strand primer which can be derived from the pre-genomic RNA putatively. Particularly, it was identified by performing mutagenesis that the conserved sequence in the RNase H enzyme was necessary for viral proliferation. In addition, the RNase H domain plays a role to synthesize a minus strand DNA as well as the plus strand DNA and to perform RNA packaging, which is identified by mutating the RNase H domain of duck HBV polymerase. But it is reported recently that duck HBV polymerase can not recognize binding region .epsilon. within the pre-genomic RNA of human HBV. Therefore, human HBV polymerase and its RNase H domain should be studied directly in addition to indirect researches by utilizing duck HBV polymerase in order to elucidate human HBV and the mechanism of its polymerase. Hitherto, the surface protein and the X protein which is necessary for the development of vaccines and for the regulation of transcription in proceeding liver cancer respectively has been studied actively. However, HBV polymerase has seldom been exploited although it can be used to develop antiviral agents. Because HBV polymerase is difficult to be separated from virus particle and to obtain sufficient amounts, especially as an active form (Radziwill, G. et al., Virology, 163: 123-132, 1988). Presently, in order to develop novel therapeutical agents for hepatitis, cell lines infected with HBV have been used for screening antiviral agents. However, effective therapeutical agents has not been yet developed, since it takes longer time and costs more for a screening method using cell lines than for screening methods using HBV polymerase or its RNase H enzyme. Recently in order to elucidate HBV, HBV polymerase and its RNase H domain have been studied as descibed above. Particularly researches for the mass production of above enzymes have been attempted by using recombinant DNA technology. The inventors of the present invention have produced a recombinant HBV polymerase which is expressed from E. coli transformant, measured its enzyme activity and filed a patent application thereof (Korean Patent Application 94-3918). The recombinant HBV polymerase was produced in E. coli as a form of fusion protein with maltose binding protein (MBP), and can be easily purified by MBP affinity column chromatography. But active HBV polymerase is difficult to be obtained massively because the polymerase can be degradaded at the C-terminus and has low purity. Foreign proteins can be obtained massively by inserting a histidine tag into the proteins by recombinant DNA technology. The nucleotide sequences encoding histidine tag is inserted into the 5'-terminus or 3'-terminus of the gene, and the histidine-tag prevents degradation of the recombinant protein so as to prepare stable enzyme. In addition, the highly active recombinant protein can be purified easily by using histidine tag affinity column as a metal chelating affinity column. In order to develop effective therapeutical agents, HBV polymerase and its RNase H enzyme have been produced by processes of the present invention. The inventors constructed expression vectors containing HBV polymerase gene with nucleotide sequences encoding a histidine tag at the C-terminus of the recombinant protein and expression vectors containing RNase H domain gene which is derived from the 3'-terminus of the HBV polymerase gene. In addition, HBV polymerase and its RNase H domain have been produced as forms of fusion protein massively in E. coli by using the expression vectors and purified easily by using amylose column and histidine tag affinity column. Thus highly active and stable HBV polymerase and its RNase H enzyme which are not degradaded can be prepared. Furthermore, the inventors have developed novel screening methods for antiviral agents by using the HBV polymerase and its RNase H domain of the present invention. SUMMARY OF THE INVENTION The object of the present invention is to provide HBV polymerase containing a histidine tag, RNase H enzyme derived from HBV polymerase and processes for preparation thereof. Particularly, the present invention provides expression vectors containing the HBV polymerase gene and process for preparing the HBV polymerase in Escherichia coli. In addition, the present invention provides expression vectors containing RNase H gene derived from human HBV polymerase gene and process for preparing the RNase H enzyme in Escherichia coli. And, the object of the present invention is to provide uses of the HBV polymerase and the RNase H enzyme for screening antiviral agents. Particularly, the present invention provides methods for screening inhibitors of the HBV polymerase and the RNase H enzyme. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a strategy for constructing the expression vector pMPH which produces the HBV polymerase containing a histidine tag. FIG. 2 depicts the HBV polymerase which has been produced and purified from E. coli NM 522/pMPH transformant by SDS-polyacrylamide gel electrophoresis. lane 1: the HBV polymerase purified primarily by amylose column lane 2: the HBV polymerase purified secondarily by histidine tag affinity column. FIG. 3 depicts a strategy for constructing the expression vector PMPRL which produces the RNase H enzyme derived from the HBV polymerase. FIG. 4 depicts a strategy for constructing the expression vector pMRH which produces the active histidine-tagged RNase H enzyme derived from human HBV polymerase. FIG. 5 depicts the RNase H enzyme which has been produced and purified from the E. coli NM 522/pMRH by SDS-polyacrylamide gel electrophoresis. lane 1: standard marker (molecular weight is 97, 68, 43 and 29 KD respectively); lane 2: crude extract of E. coli transformant lane 3: the RNase H enzyme purified primarily by amylose column lane 4: the RNase H enzyme purified secondarily by histidine tag affinity column. FIG. 6 depicts the RNase H enzyme which has been produced and purified from the E. coli NM522/pMRH transformant by western blotting analysis. A: a result using anti-maltose binding protein B: a result using antibody against histidine tag (.sup.MRGS HIS) Each lane is explained on the above FIG. 5 FIG. 7 depicts RNA/DNA complex used in assaying the RNase H activity of the present invention. FIG. 8 represents the results comparing RNase H activities of following samples, (i) maltose binding protein, (ii) crude extract of the E. coli transformant cultured, (iii) the RNase H expressed and purified by using the expression vector pMPRL and amylose column, (iv) the RNase H expressed and purified by using the expression vector pMRH and amylose column, histidine affinity column, (v) reverse transcriptase of Moloney murine Leukemia Virus. FIG. 9 represents the increase of the RNase H activity according to the amount of the RNase H enzyme. FIG. 10 represents the increase of the RNase H activity according to the reaction period of the RNase H enzyme. FIG. 11 represents the variation of the RNase H activity according to the reaction temperature. FIG. 12 represents the variation of the RNase H activity according to the pH of the reaction solution. FIG. 13 represents the variation of the RNase H activity according to the concentration of potassium chloride. FIG. 14 represents the variation of the RNase H activity according to the concentration of magnesium ion. FIG. 15 represents the variation of the RNase H activity according to the concentration of manganese ion. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides HBV polymerase containing a histidine tag which is prepared by inserting nucleotide sequences of the histidine tag into the end of HBV polymerase gene. Since the HBV polymerase containing a histidine tag is stable and can be easily purified, its activity of reverse transcriptase and the like can be measured properly. By using site-specific insertion mutagenesis and so on, nucleotide sequences of histidine tag can be inserted into 5'-terminus or 3'-terminus of the HBV polymerase gene. Particularly, the present invention has exploited the expression vector already established (Korean Patent Application 94-3918) which produces the HBV polymerase fused with maltose binding protein (MBP). And nucleotide sequence of 6 histidine residues is inserted into the 3'-terminus of HBV polymerase gene. Especially since the histidine codons are inserted continuously right before the stop codon, the open reading frame (ORF) of the HBV polymerase gene is setted exactly. The histidine tag can be inserted into the C-terminus of the polymerase, which maintains the enzyme activity. As a result, the expression vector pMPH has been constructed, which can produce the HBV polymerase fused with maltose binding protein and histidine tag (see FIG. 1). To express the recombinant polymerase, microorganism is transformed with the expression vector pMPH so as to prepare transformant. The microorganism mentioned above contains all kinds of Escherichia coli which is suitable for the expression of recombinant proteins. Particularly, E. coli NM522 strain was transformed with the expression vector pMPH and the transformant has been deposited with Korean Culture Center of Microorganism, Seoul, Korea, on Jul. 19, 1996 (Accession number: KCCM-10084). The present invention provides a process for preparing the recombinant HBV polymerase massively. E. coli transformant containing the expression vector is induced to express the recombinant protein and disrupted to obtain crude extract, then the HBV polymerase is purified by using histidine tag affinity column chromatography and other chromatographies. Precisely, since the E. coli transformant containing the expression vector pMPH produces the recombinant HBV polymerase fused with MBP at the N-terminus, the HBV polymerase is purified as a form of fusion protein by using amylose resin column. And the histidine tagged polymerase only can be obtained separating MBP by treating protease factor Xa and the like. In additon, the histidine tagged HBV polymerase is purified highly and conveniently by performing the metal chelating affinity column as histidine tag affinity column. The histidine tag maintains enzyme activity of the recombinant HBV polymerase during the purification process since it prevents protein degradation as well as facilitates the purification process of the HBV polymerase. The present invention provides the RNase H enzyme derived from HBV polymerase. Since human HBV polymerase has a RNase H domain with enzyme activity at the N-terminus, the RNase H domain of the present invention is prepared by inserting 3'-terminus of the HBV polymerase gene into a expression vector and inducing E. coli transformant. The present invention provides expression vectors which produces the RNase H enzyme fused with MBP to prepare the RNase H enzyme derived from the HBV polymerase. Precisely, the RNase H subdomain gene of the HBV polymerase can be obtained by performing polymerase chain reaction (PCR) which utilizes oligonucleotides of SEQ ID. NO: 2 and SEQ ID. NO: 3 as primers (see Sequence Listing) and the expression vecor pMPLX already established as a template. The expression vector pMPLX can produce the HBV polymerase as a form fused with MBP (Korean Patent Application 94-3918). RNase H enzyme gene obtained above has been inserted into the plasmid vector pMAL-c2 to construct the expression vector pMPRL (see FIG. 3). Particularly, E. coli NM522 strain was transformed with the expression vector pMRH and the transformant has been deposited with Korean Culture Center of Microorganism, Seoul, Korea, on Nov. 29, 1996 (Accession number: KCCM-10092). In addition, the present invention provides expression vectors which produces RNase H enzyme fused with MBP and histidine tag in order to prepare the RNase H enzyme derived from the HBV polymerase. Precisely, the gene fragment of the HBV polymerase containing nucleotide sequences of histidine tag is obtained from the expression vector pMPH and inserted into the expression vector pMPRL to construct the expression vector pMRH (see FIG. 4). Particularly, E. coli NM522 strain was transformed with the expression vector pMRH and the transformant has been deposited with Korean Culture Center of Microorganism, Seoul, Korea, on Nov. 11, 1996 (Accession number: KCCM-10091). The present invention provides a process for preparing the RNase H enzyme derived from the HBV polymerase by utilizing the expression vectors and the transformants describe above. Precisely in order to purify the RNase H domain of the HBV polymerase, E. coli transformant containing the expression vector is induced for the protein expression, disrupted to obtain crude extracts, then RNase H enzyme as a form of fusion protein is purified by using amylose resin and maltose-containing buffer. And the histidine tagged RNase H enzyme only can be obtained separating MBP by treating protease factor Xa and the like. And the histidine tagged RNase H enzyme is purified higherly by performing histidine tag affinity column chromatography as the same process described above. Molecular weights and purity of the HBV polymerase and the RNase H enzyme purified above have been determined by using SDS-polyacrylamide gel electrophoresis and western blotting. As a result, the HBV polymerase and the RNase H enzyme of the present invention is identified to be intact forms which has not been degraded (see FIG. 2, FIG. 5 and FIG. 6). And reverse transcriptase activity in the HBV polymerase of the present invention has been examined. As a result, the recombinant polymerase with MBP and histidine tag has shown higher activity of reverse transcriptase than the polymerase without histidine tag. In detail, activities of DNA dependent DNA polymerase (DDDP) and RNA dependent DNA polymerase (RDDP) have been 19 times higher in histidine tagged form than those in intact form (see Table 1). Precisely, in order to investigate RNA degradation by the RNase H activity, RNA/DNA complex is used as a substrate for the enzyme reaction. As a DNA template for preparing RNA/DNA complex, the plasmid pBS-oligo derived from the plamid pBS is selected, digested within restriction site SmaI, 102 nucleotides downstream from T7 promoter and then in vitro translation has been performed by using T7 RNA polymerase, radioactive nucleotides and so on. And RNA of 102 nucleotides is obtained by using QIAquick nucleotide removal kit, QIAGEN and synthetic DNA oligonucleotide (43-mer) of SEQ ID NO: 4 is added to prepare RNA/DNA complex which is shown in FIG. 7 (see Sequence Listing). In order to measure the RNase H enzyme activity, radioactive RNA/DNA complex is reacted with the RNase H enzyme and the radioactivity in the supernatant of the reaction mixture is measured by using scintillation cocktail and the like. At that time maltose binding protein as a contrast sample, crude extract fraction, commercially available reverse transcriptase of Moloney murine Leukemia Virus as a comparative sample and so on are utilized. As a result, RNase H enzyme of the present invention is more active than that of reverse transcriptase of Moloney murine Leukemia virus, approximately 90% activity (see FIG. 8). In order to examine the enzymatic properties of the RNase H domain, the RNase H activity is measured in various reaction conditions by using proper buffers and radioactive RNA/DNA complex. As results, the enzyme activity of the RNase H domain increases according to the enzyme amount (see FIG. 9) and takes about 3 hours of the reaction period to be shown (see FIG. 10). And preferably the temperature range is 32-42.degree. C. for the enzymatic reaction (see FIG. 11) and pH range is broad comparatively such as 7.5-8.8 (see FIG. 12). And preferably the reaction mixture for the enzymatic reaction should have 20-100 mM range of KCl concentration (see FIG. 13), 4-8 mM range of magnesium concentration (see FIG. 14), and 4-12 mM range of manganese concentration (see FIG. 15). More preferably, for the enzymatic reaction of the RNase H optimun temperature is 37.degree. C., optimum pH is 7.9, optimum NaCl concentration is 40 mM, magnesium ion is 4 mM and manganese ion is 8 mM. And the present invention provides uses of the HBV polymerase and the RNase H enzyme derived from the HBV polymerase for screening antiviral agents. In order to select HBV inhibitors working at the multiplication stage of HBV by using the HBV polymerase, (a) the HBV polymerase is reacted with homopolymer template, radioactive nucleotide and antiviral agent, (b) the reaction solution of (a) stage is adsorpted onto anion adsorption filter and dried, (c) the radioactivity of the adsorbent filter is measured by using scintillation cocktail and, (d) the results of (c) stage is compared with those of comparative sample which does not contain a antiviral agent in the reaction mixture and used to calculate the inhibitory effects of HBV multiplication. Then poly(da)/oligo(dT).sub.12-18 is used as homopolymer template for DDDP activity and poly(rA)/oligo(dT).sub.12-18 for RDDP activity preferably and DE-81 anion adsorbent filter is used preferably. In addition, in order to select antiviral agents by using the RNase H enzyme derived from the HBV polymerase, at first, enzyme substrates should be prepared by the process described below and then the radioactivity of the substrate should be measured. In order to select HBV inhibitors working at the multiplication stage of HBV by using the RNase H domain of the HBV polymerase, (a) the RNase H enzyme is reacted with the reaction substrate and antiviral agent, (b) ammonium acetate is added to stop the reaction of (a) stage and precipitated by adding ethanol and centrifuging, (c) the radioactivity of the supernatant of the precipitate is measured and, (d) the results of (c) stage is compared with those of comparative sample which does not contain an antiviral agent in the reaction mixture and used to calculate the inhibitory effects of HBV multiplication. Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modification and improvements within the spirit and scope of the present invention. |
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