Expression of a hepatitis C virus NS3 protease-NS4A fusion protein in Escherichia coli.
Both the NS3 protease and the NS4A protein are required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. The NS3 protease domain was fused at its C-terminal end with full length NS4A and expressed in Escherichia coli. This protein (NS3 delta-NS4A) was purified to apparent homogeneity after refolding from extracts recovered from inclusion bodies. During the expression and purification process, NS3 delta-NS4A was not auto-processed in either a cis or trans manner at NS3/NS4A junction site. When the kcat/K(m) values and thermostability of NS3 delta-NS4A were compared with those for maltose binding protein-NS3 fusion protein (MBP-NS3), which contains only NS3 region, the single-chain NS3 delta-NS4A showed enhanced proteolytic activities and thermostability.
Inoue
H
H
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Ibaraki, Japan. inoue_h@yamanouchi.co.jp
Sakashita
H
H
Shimizu
Y
Y
Yamaji
K
K
Yokota
T
T
Sudo
K
K
Shigeta
S
S
Shimotohno
K
K
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C461599:maltose transport system, E coli