Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli.
We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.
Bablanian
G M
GM
USDA, ARS, NAA, Plum Island Animal Disease Center, Greenport, New York 11944.
Grubman
M J
MJ
D000086042:3C Viral Proteases; D005537:Aphthovirus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D011994:Recombinant Proteins; D014764:Viral Proteins; D008715:Methionine; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases