title: Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide.
abstract: We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan; Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunacho, Chuo-ku, Tokyo 103-0024, Japan. Electronic address: t.akuta@kyokutoseiyaku.co.jp.
authors.forename: Teruo
authors.initials: T
authors.lastname: Akuta
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Takane
authors.initials: T
authors.lastname: Kikuchi-Ueda
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan; Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunacho, Chuo-ku, Tokyo 103-0024, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Imaizumi
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. Electronic address: onoy@med.teikyo-u.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ono
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D019089:Stem Cell Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015854:Up-Regulation
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D019089:Stem Cell Factor; C000611742:glutathione persulfide; C116840:cysteine persulfide; D005978:Glutathione; D003545:Cysteine
keywords: Affinity tag-based protein purification; Escherichia coli; Human stem cell factor; Persulfide; Soluble protein expression