Expression and purification of an active, full-length hepatitis C viral NS4A.
The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.
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S H
SH
Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, Kyungbuk, 790-784, Korea.
Kim
J E
JE
Rho
J
J
Hahm
B
B
Lee
T G
TG
Kim
E E
EE
Cho
J M
JM
Jang
S K
SK
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005990:Glycerol / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D003067:Coenzymes; D010450:Endopeptidases; D029968:Escherichia coli Proteins; D005990:Glycerol; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C461599:maltose transport system, E coli