Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus.
The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.
Piccone
M E
ME
Plum Island Animal Disease Center, Greenport, NY 11944, USA.
Sira
S
S
Zellner
M
M
Grubman
M J
MJ
D005537:Aphthovirus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D012315:RNA Caps; D011994:Recombinant Proteins; D014764:Viral Proteins; C069883:initiation factor 4F component p220; D010450:Endopeptidases; C088747:leader proteinase, foot-and-mouth disease virus