Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli.
The serine protease domain of HCV comprising amino acids 1027-1218 (deltaNS3) was expressed in E. coli with a His tag at its N-terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (approximately 3 mg/l of cultured cells). The deltaNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with kcat/Km = 160 x 10(-3) min(-1) microM(-1) in the presence of NS4A peptide. Our deltaNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The deltaNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203-2506 amino acids) that was synthesized by in vitro transcription and translation system.
Vishnuvardhan
D
D
National Institute of Bioscience and Human Technology, AIST, Tsukuba, Ibaraki, Japan.
Kakiuchi
N
N
Urvil
P T
PT
Shimotohno
K
K
Kumar
P K
PK
Nishikawa
S
S
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020365:RNA Helicases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017931:DNA Primers; D010446:Peptide Fragments; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases