title: [Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin].
abstract: The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.
authors.affiliation: Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, School of Life Science, Anhui University, Hefei 230039, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.forename: Hui
authors.initials: H
authors.lastname: Peng
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.forename: Yongzhong
authors.initials: Y
authors.lastname: Wang
authors.forename: Yazhong
authors.initials: Y
authors.lastname: Xiao
mesh_terms: D000891:Anti-Infective Agents, Local / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008248:Lysostaphin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D000891:Anti-Infective Agents, Local; D050505:Mutant Proteins; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D011092:Polyethylene Glycols; D008248:Lysostaphin; D003545:Cysteine