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1.发明授权METHOD FOR ASSAYING IN VITRO THE PROTEOLYTIC ACTIVITY OF POLYPEPTIDES HAVING HCV NS3 PROTEASE ACTIVITY AND PEPTIDE SUBSTRATES TO BE USED IN THIS METHOD. 无效丙型肝炎病毒NS3蛋白酶多肽具有活性和肽底体外测试蛋白水解活性的方法此过程中使用
公开(公告)号：EP0846164B1
公开(公告)日：2005-03-02
申请号：EP96926574.3
申请日：1996-08-20
发明人： STEINKÜHLER, Christian , PESSI, Antonello , BIANCHI, Elisabetta , TALIANI, Marina , TOMEI, Licia , URBANI, Andrea , DE FRANCESCO, Raffaele , NARJES, Frank
CPC分类号： C07K14/005 , C12N9/506 , C12N2770/24222
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2.发明公开METHODOLOGY TO PRODUCE, PURIFY AND ASSAY POLYPEPTIDES WITH THE PROTEOLYTIC ACTIVITY OF THE HCV NS3 PROTEASE 无效丙型肝炎病毒NS3蛋白酶多肽具有活性和肽底体外测试蛋白水解活性的方法此过程中使用
公开(公告)号：EP0846164A2
公开(公告)日：1998-06-10
申请号：EP96926574.0
申请日：1996-08-20
发明人： STEINKÜHLER, Christian , PESSI, Antonello , BIANCHI, Elisabetta , TALIANI, Marina , TOMEI, Licia , URBANI, Andrea , DE FRANCESCO, Raffaele , NARJES, Frank
CPC分类号： C07K14/005 , C12N9/506 , C12N2770/24222
摘要： The process according to the present invention allows expression and isolation of polypeptides with the proteolytic activity of HCV NS3 protease in a pure, catalytically active form, and in amounts that are sufficient for discovery of NS3 protease inhibitors and for determination of the three-dimensional structure of the NS3 protease. A further subject of the present invention is a procedure that defines the chemical and physical conditions necessary for completion of the proteolytic activity of the above polypeptides. The invention further comprises new compositions of matter (expression vectors) containing nucleotide sequences capable of expressing the above mentioned polypeptides in culture cells. Finally, new compounds of matter are defined, suitable to measure the above proteolytic activity, and useful to develop NS3 protease inhibitors and therefore therapeutic agents for use against HCV. The figure shows the kinetic parameters of HCV NS3 protease using the S3 depsipeptide substrate (SEQ ID NO:45).
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3.发明授权METHOD FOR REPRODUCING IN VITRO THE PROTEOLYTIC ACTIVITY OF THE NS3 PROTEASE OF HEPATITIS C VIRUS (HCV) 失效VERFAHRENM FOR体外产生蛋白水解的影响丙型肝炎病毒NS3学院学报（HCV）
公开(公告)号：EP0746333B1
公开(公告)日：1999-05-06
申请号：EP95909937.5
申请日：1995-02-14
CPC分类号： C07K14/005 , C12N2770/24222 , G01N33/573 , G01N33/5767
摘要： This is a method for reproducing in vitro the serine protease activity associated with the HCV NS3 protein, that comprises the use both of sequences contained in NS3 and sequences contained in NS4A. This method takes advantage of the ability of the HCV NS4A protein, or sequences contained therein, to act as a cofactor of the serine protease activity or more generally of the enzymatic activities associated with NS3. Optimal serine protease activity is obtained when NS4A is present in a molar ratio of at least 1:1 with NS3. NS3 and NS4A can also be incorporated in the reaction mixture as NS3-NS4A precursor, as this precursor will generate, by means of an autoproteolytic event, equimolar amounts of NS3 and NS4A. It is also possible to mutate the cleavage site between NS3 and NS4A in a precursor, so that NS4A remains covalently bonded to NS3. The sequences that do not influence the proteolytic activity of NS3 can subsequently be removed from said non-proteolyzable precursor. The invention also relates to a composition of matter that comprises sequences contained in NS3 and NS4A, and to the use of these compositions for the setup of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS3. The figure shows plasmidic vectors used in the method to activate HCV NS3 protease in cultivated cells and in vitro.
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4.发明公开METHOD FOR REPRODUCING IN VITRO THE RNA-DEPENDENT RNA POLYMERASE AND TERMINAL NUCLEOTIDYL TRANSFERASE ACTIVITIES ENCODED BY HEPATITIS C VIRUS (HCV) 无效METHOD FOR RNA依赖性RNA聚合酶和丙型肝炎病毒末端核苷酸基转移酶活性的体外繁殖（HCV）
公开(公告)号：EP0842276A1
公开(公告)日：1998-05-20
申请号：EP96916289.0
申请日：1996-05-24
CPC分类号： C12N9/1241 , C12N9/127 , C12N2799/026
摘要： This is a method for reproducing in vitro the RNA-dependent RNA polymerase activity associated with hepatitis C virus. The method is characterized in that sequences contained in NS5B are used in the reaction mixture. The terminal nucleotidyl transferase activity, a further property of the NS5B protein, can also be reproduced using this method. The method takes advantage of the fact that the NS5B protein, either purified to apparent homogeneity or present in extracts of overproducing organisms, can catalyse the addition of ribonucleotides to the 3'-termini of exogenous or endogenous RNA molecules. The invention also relates to a composition of matter that comprises sequences contained in NS5B, and to the use of these compositions for the set up of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS5B. The figure shows plasmids used in the method to produce hepatitis C virus RNA-dependent RNA polymerase and terminal nucleotidyl transferase in cultivated eukaryotic and prokaryotic cells.
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5.发明公开METHOD FOR REPRODUCING IN VITRO THE PROTEOLYTIC ACTIVITY OF THE NS3 PROTEASE OF HEPATITIS C VIRUS (HCV) 失效VERFAHRENM FOR体外产生蛋白水解的影响丙型肝炎病毒NS3学院学报（HCV）
公开(公告)号：EP0746333A1
公开(公告)日：1996-12-11
申请号：EP95909937.0
申请日：1995-02-14
CPC分类号： C07K14/005 , C12N2770/24222 , G01N33/573 , G01N33/5767
摘要： This is a method for reproducing in vitro the serine protease activity associated with the HCV NS3 protein, that comprises the use both of sequences contained in NS3 and sequences contained in NS4A. This method takes advantage of the ability of the HCV NS4A protein, or sequences contained therein, to act as a cofactor of the serine protease activity or more generally of the enzymatic activities associated with NS3. Optimal serine protease activity is obtained when NS4A is present in a molar ratio of at least 1:1 with NS3. NS3 and NS4A can also be incorporated in the reaction mixture as NS3-NS4A precursor, as this precursor will generate, by means of an autoproteolytic event, equimolar amounts of NS3 and NS4A. It is also possible to mutate the cleavage site between NS3 and NS4A in a precursor, so that NS4A remains covalently bonded to NS3. The sequences that do not influence the proteolytic activity of NS3 can subsequently be removed from said non-proteolyzable precursor. The invention also relates to a composition of matter that comprises sequences contained in NS3 and NS4A, and to the use of these compositions for the setup of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS3. The figure shows plasmidic vectors used in the method to activate HCV NS3 protease in cultivated cells and in vitro.
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6.发明授权METHOD FOR REPRODUCING IN VITRO THE RNA-DEPENDENT RNA POLYMERASE AND TERMINAL NUCLEOTIDYL TRANSFERASE ACTIVITIES ENCODED BY HEPATITIS C VIRUS (HCV) 无效METHOD FOR RNA依赖性RNA聚合酶和丙型肝炎病毒末端核苷酸基转移酶活性的体外繁殖（HCV）
公开(公告)号：EP0842276B1
公开(公告)日：2004-01-21
申请号：EP96916289.0
申请日：1996-05-24
IPC分类号： C12N15/54 , C12N9/12 , C12Q1/48 , G01N33/573
CPC分类号： C12N9/1241 , C12N9/127 , C12N2799/026
摘要： This is a method for reproducing in vitro the RNA-dependent RNA polymerase activity associated with hepatitis C virus. The method is characterized in that sequences contained in NS5B are used in the reaction mixture. The terminal nucleotidyl transferase activity, a further property of the NS5B protein, can also be reproduced using this method. The method takes advantage of the fact that the NS5B protein, either purified to apparent homogeneity or present in extracts of overproducing organisms, can catalyse the addition of ribonucleotides to the 3'-termini of exogenous or endogenous RNA molecules. The invention also relates to a composition of matter that comprises sequences contained in NS5B, and to the use of these compositions for the set up of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS5B. The figure shows plasmids used in the method to produce hepatitis C virus RNA-dependent RNA polymerase and terminal nucleotidyl transferase in cultivated eukaryotic and prokaryotic cells.
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7.发明公开SOLUBLE POLYPEPTIDES WITH ACTIVITY OF THE NS3 SERINE PROTEASE OF HEPATITIS C VIRUS, AND PROCESS FOR THEIR PREPARATION AND ISOLATION 失效丙型肝炎病毒的NS3丝氨酸蛋白酶为他们的制备和分离的活性，AND METHOD可溶性多肽。
公开(公告)号：EP0950094A1
公开(公告)日：1999-10-20
申请号：EP97942190.0
申请日：1997-09-17
发明人： DE FRANCESCO, Raffaele , TRAMONTANO, Anna , TOMEI, Licia , NARDI, Maria, Chiara , STEINKÜHLER, Christian , URBANI, Andrea , VITALE, Rosa, Letizia , COLLOCA, Stefano
CPC分类号： C12N9/506
摘要： The present invention relates to serine protease NS3 of hepatitis C virus, and in particular to the observation that the NS3 serine protease domain, in its native conformation, binds a Zn2+ ion and that bivalent metallic ions are necessary to the structural integrity of the protein and to the activity of the enzyme. The present invention further relates to recombinant polypeptides which comprise sequences of the NS3 protease and are characterised by a tail of at least three lysines at their C-terminal ends, to increase its solubility. A further subject of the present invention is a new process which allows the expression of said polypeptides, as metalloproteins, with the proteolytic activity of the HCV NS3 protease, in a soluble form and in a quantity sufficient to allow research to identify inhibitors and to determine the three-dimensional structure of the NS3 protease. Figure 4 shows the effects of the zinc ion on the production of the HCV NS3 protease as a soluble protein in E. Coli in a minimum culture medium.
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