Engineering a novel endopeptidase based on SARS 3CLpro

A 3C-like protease (3CL pro ) 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↓(Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CL pro (T25G) with an expanded S1...

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Bibliographic Details
Published in:BioTechniques 2018-04, Vol.47 (6), p.1029-1032
Main Authors: Chih-Jung, Kuo, Yan-Ping, Shih, Kan, Daphne, Po-Huang, Liang
Format: Article
Language:English
Subjects:
3CL protease
endopeptidase
parallel cloning
SARS-CoV
Short Technical Reports
tag cleavage
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Summary:A 3C-like protease (3CL pro ) 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↓(Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CL pro (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↓Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. Pst I 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.
ISSN:0736-6205
1940-9818
DOI:10.2144/000113303