Expression and purification of recombinant 3C proteinase of Coxsackievirus B3.
We have cloned various lengths of coxsackievirus B3 cDNA encompassing the region encoding the 3C proteinase, which is essential to the viral replication cycle. Such viral cDNAs were fused in frame to the 5'terminal portion of the lacZ' gene carried on the vector pUC118 to express mature 3C proteinase in Escherichia coli. In the E. coli cells containing pCXB108 or pCXB117, constructed for this study, a large amount of 23-kDa protein was synthesized in the presence of IPTG. This protein was purified and was shown to be intact 3C proteinase. These data suggest that 3C proteinase, expressed as a part of a fusion protein, was active in E. coli and released itself from the precursor fusion protein by autocatalytic cleavage.
Miyashita
K
K
Central Research Laboratories, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan.
Kusumi
M
M
Utsumi
R
R
Komano
T
T
Satoh
N
N
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D029822:Enterovirus B, Human / Q000201:enzymology* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004279:DNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases