The expression and purification of human rhinovirus protease 3C.
Human rhinovirus type 14 protease 3C was expressed as a soluble and active protein in Escherichia coli. The protease was purified by a cationic-exchange step followed by gel filtration on a TSK 3000 column. The final yield of purified protease was in the range 0.5-1.0 mg/l culture grown to A550 = 1.0. Sequence analysis revealed that greater than 90% of the N-terminal residues were methionine. The enzyme activity of the purified protease was measured by cleavage of a synthetic peptide representing a predicted Gln/Gly viral polyprotein cleavage site. A mutant protease (Cys146----Ser) was produced and purified in the same way. The yield of mutant protease 3C was approximately 150 micrograms/l from a culture grown to A550 = 1.0. This mutant protease 3C did not cleave the synthetic peptide substrate.
Knott
J A
JA
Genetics Department, Glaxo Group Research, Greenford, Middlesex, England.
Orr
D C
DC
Montgomery
D S
DS
Sullivan
C A
CA
Weston
A
A
D000086042:3C Viral Proteases; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins
D000596:Amino Acids; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases