Abstract: The special connection approaches of linear ubiquitin chain have been elucidated in recent years. Currently, the types of commercial linear ubiquitin chain specific antibodies are limited and their specificity still needs to be improved. RHD1 is a papain-like deubiquitinating enzyme that specifically recognizes and cleaves linear ubiquitin chains. The mutation at C13 in its enzymatic activity site results in the loss of deubiquitination function for the RHD1. Meanwhile, the RHD1 with this mutation still exhibits low binding activity towards linear ubiquitin chain. On the basis of this mutant, we optimized the mutations at interaction interface between RHD1 and Di-Ub (M1-linked) in order to enhance the specific binding activity of RHD1. In this study, we utilized the structural model of RHD1 and Di-Ub (M1-linked) complex to analyze the physical and chemical properties of all amino acids at the interaction interface and subsequently designed a RHD1 mutant library. The site-mutations in RHD1 variant polypeptides were generated by using overlap extension PCR method, and all recombinant variant proteins were expressed by using E. coli BL21 (DE3) strain. By employing the enzyme-linked immune sorbent assay (ELISA ) method for screening, we successfully identified a RHD1 variant harboring C13A and A92T mutations, which showed significantly increased affinity to Tetra-Ub (M1-linked). This RHD1 variant holds great potential as a valuable tool for studying linear ubiquitin chains, and exhibits promising application in clinical diagnostics and the development of related therapeutic agents.
Key words: RHD1 deubiquitinating enzymes; Tetra-Ub (M1-linked); site-specific mutation; prokaryotic expression; ELISA
（Acta Laser Biology Sinica, 2024, 33(5): 439-449）