title: Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.
abstract: Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.
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. Electronic address: takane@med.teikyo-u.ac.jp.
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 Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oshikane
authors.affiliation: Department of Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Nakaki
authors.affiliation: Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okada
authors.affiliation: Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan.
authors.forename: Sara
authors.initials: S
authors.lastname: Sultana
authors.affiliation: Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kiyokawa
authors.affiliation: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ono
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D019089:Stem Cell Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D013879:Thioredoxins; D001120:Arginine
keywords: Affinity tag-based protein purification; Escherichia coli; Human stem cell factor; Soluble protein expression; l-Arginine