Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus.
Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.
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K W
KW
Department of Pathology, University of Cambridge, UK. kwt@mole.bio.cam.ac.uk
Cavanagh
D
D
Brown
T D
TD
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006632:Histamine / Q000737:chemistry / Q000235:genetics*; D001351:Infectious bronchitis virus / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D006632:Histamine; D003546:Cysteine Endopeptidases; C000706208:3C-like proteinase, Avian infectious bronchitis virus; D000086782:Coronavirus 3C Proteases