Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli.
A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.
Takahara
Y
Y
Genetic Engineering Section, Sagami Chemical Research Center, Kanagawa, Japan.
Ando
N
N
Kohara
M
M
Hagino-Yamagishi
K
K
Nomoto
A
A
Itoh
H
H
Numao
N
N
Kondo
K
K
D000086042:3C Viral Proteases; D002213:Capsid / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014170:Transformation, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D011498:Protein Precursors; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases