Engineering a novel endopeptidase based on SARS 3CL(pro).
A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln [downward arrow]Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. PstI was chosen as a 5' cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL(pro) (T25G) was found to have a 3-fold improvement over TEV(pro) in tag cleavage at each respective preferred cleavage site.
Kuo
Chih-Jung
CJ
Institute of Biological Chemistry, Academia Sinica and Core Facility of Recombinant Protein Production, Taipei, Taiwan.
Shih
Yan-Ping
YP
Kan
Daphne
D
Liang
Po-Huang
PH
D000595:Amino Acid Sequence; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C099488:TEV protease; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases