A Combinatorial Strategy for HRV 3C Protease Engineering to Achieve the N-terminal Free Cleavage

Human rhinovirus 3C protease (HRV 3C-P) has a high specificity against the substrate of LEVLFQ{downarrow}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 3C-P, 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 3C-P 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{downarrow}M (kcat/KM= 3.72 {+/-} 0.04 mM-1{middle dot}s-1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2 site, but with an expanded P1 specificity. Structural simulation of C3 indicated a reconstructed S1 binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3C-P, also provide new tools for other enzyme engineering. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/574269v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b891eborg.highwire.dtl.DTLVardef@174151dorg.highwire.dtl.DTLVardef@1656f4org.highwire.dtl.DTLVardef@a6c1bb_HPS_FORMAT_FIGEXP M_FIG C_FIG