title: Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity inE. colivia Mutation of Free Cysteine Residues.
abstract: Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields inE. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 inE. coliwhereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.
authors.affiliation: Microbiology Institute of Shaanxi, Xi'an 710043, China.
authors.forename: Kun
authors.identifier: 0000-0002-1448-1373
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Microbiology Institute of Shaanxi, Xi'an 710043, China.
authors.forename: Yuejuan
authors.identifier: 0000-0001-8120-2031
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Microbiology Institute of Shaanxi, Xi'an 710043, China.
authors.forename: Jing
authors.identifier: 0000-0001-5984-6834
authors.initials: J
authors.lastname: Zi
authors.affiliation: State Key Laboratory of Cancer Biology, Department of Biopharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Xiaochang
authors.identifier: 0000-0001-9718-4659
authors.initials: X
authors.lastname: Xue
authors.affiliation: Microbiology Institute of Shaanxi, Xi'an 710043, China.
authors.forename: Yi
authors.identifier: 0000-0002-0275-9374
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D004249:DNA Damage; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008544:Melanocytes / Q000166:cytology; D008546:Melanoma, Experimental; D009154:Mutation; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis; D000072105:Superoxide Dismutase-1 / Q000096:biosynthesis*
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D000072105:Superoxide Dismutase-1; D003545:Cysteine