A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage.

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1' site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1' specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1' specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (k

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Copyright © 2024. Published by Elsevier B.V.

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Declaration of competing interest The authors declare no conflict of interest.