Evaluation of the disulfide bond positions in recombinant Gaussia luciferase expressed in Escherichia coli cells by site-directed mutagenesis.

Abstract

Gaussia luciferase (GLase) is a secreted enzyme composed of 168 amino acids, including 10 cysteine residues, and catalyzes the oxidation of coelenterazine to emit light. To evaluate the disulfide bond positions in GLase, we generated 10 cysteine-to-serine substituted GLase genes, in which each cysteine residue was replaced with a serine residue (C52S, C56S, C59S, C65S, C77S, C120S, C123S, C127S, C136S, and C148S), using site-directed mutagenesis. In both bacterial and mammalian expression systems, four disulfide bonds formed between eight cysteine residues (C52, C56, C65, C77, C123, C127, C136, and C148) were found to be essential for luminescence activity. In bacterial cells, the single mutants C59S and C120S, as well as the double mutant C59S/C120S, exhibited luminescence activities of 258%, 2.8%, and 42.8%, respectively, relative to wild-type GLase (100%). Notably, all three mutants could be efficiently refolded by dialysis after treatment with 2-mercaptoethanol. In mammalian cells, only the double mutant C59S/C120S was secreted and showed luminescence activity of 11% in the culture medium, relative to wild-type GLase (100%). By integrating previously reported NMR-based structural data of recombinant GLase purified from bacterial cells with our experimental findings, we conclude that GLase contains five disulfide bonds: C52-C127, C56-C123, C59-C120, C65-C77, and C136-C148, which are consistent with those reported in PDB ID: 7D2O.