Engineering a novel endopeptidase based on SARS 3CL(pro)

Abstract

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.

Figure 1.. Structural basis for T25 mutation.

Predicted structural model of SARS-CoV 3CL^pro with a modified peptide containing Met at P1′ (Thr-Ser-Ala-Val-Leu-Gln-Met-Phe-Arg-Lys), based on the crystal structure of SARS-CoV 3CL^pro H41A mutant in complex with a peptide (PDB entry: 2Q6G).

Figure 2.. Characterization of the wild-type and mutant 3CL^pro.

(A) Activities of wild-type, T25G, T25S, and C-terminal His-tagged T25G 3CL^pro against a fluorogenic substrate Dabcyl-KTSAVLQSGFRKME-Edans (upper panel). The cleavage rate by T25G 3CL^pro (83.5 µM/min) was remarkably higher than the rate of the wild-type (6.8 µM/min) against a peptide substrate SAVLQ↓MGFRK containing Met at P1′ (lower panel). (B) Substrate specificity of T25G 3CL^pro at P1′ site. Activities of wild-type and T25G 3CL^pro using 10 peptides SAVLQXGFRK (X = Glu, Phe, Gly, His, Lys, Leu, Met, Pro, Ser, and Trp) as substrates.

Figure 3.. Expression of the UPPs fusion protein in E. coli and tag cleavage by the wild-type and mutant 3CL^pro.

(A) The strategy of using PstI as a 5′ cloning site (CTGCAG) that is part of the sequence GCGGTGCTGCAG encoding the protease recognition site is illustrated. (B) 5.4 µM UPPs fusion protein before treatment (lane 1) and after treatment with 0.1 µM wild-type (lane 2), T25G (lane 3), and T25S 3CL^pro (lane 4) for 90 min at 37°C are shown. Only T25G can efficiently cleave the fusion protein to generate tag-free UPPs.