Effects of removing a highly conserved disulfide bond in ubiquitin-associated domain of human HOIP on biochemical characteristics

Abstract

Disulfide bond formed between the cysteine pairs plays a key role in maintaining the integrity of the protein structure and function. The ubiquitin-associated (UBA) domain of human HOIP contains three cysteine residues, Cys504, Cys551, and Cys572. Disulfide bonds formed by Cys504 and Cys551 residues are highly conserved, but the effect of disulfide bonds on the biochemical characteristics of UBA has not been elucidated. In addition, due to the presence of isolated Cys572, inactive inclusion bodies may be formed during protein expression or trigger protein aggregation during protein purification. In this study, the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of UBA domain. Introduced three mutants (UBAC551A, UBAC572A and UBAC551,572A) determined the effects of disulfide bonds on the biochemical characteristics of UBA. Circular dichroism and analytical size exclusion chromatography results showed that the target proteins obtained by co-expression could be folded correctly and had biological activity. Both thermal-induced and urea-induced results demonstrated that the elimination of disulfide bonds would significantly reduce the stability of UBA. Fluorescence spectroscopy result showed that the elimination of disulfide bonds slightly increases the binding affinity of UBA to ligands. In summary, soluble, stable and active UBA domain and its mutants were prepared by co-expression system, which will further contribute to the structural and functional research of UBA.