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 Teruo Akuta a,b,⇑ , Takane Kikuchi-Ueda a,⇑ , Keitaro Imaizumi a,b , Hiroyuki Oshikane c , Toshio Nakaki c , Yoko Okada d , Sara Sultana d , Kenichiro Kobayashi d , Nobutaka Kiyokawa d , Yasuo Ono a a Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan b Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunacho, Chuo-ku, Tokyo 103-0024, Japan c Department of Pharmacology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan d 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 article info Article history: Received 28 August 2014 and in revised form 19 September 2014 Available online 5 October 2014 Keywords: Human stem cell factor Escherichia coli Soluble protein expression Affinity tag-based protein purification L-Arginine abstract Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the reg- ulation 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 homodi- mer 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) harbor- ing 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 0.1 M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5 M L-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the pro- tein 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. Ó 2014 Elsevier Inc. All rights reserved. Introduction Stem cell factor (SCF) is a dimeric molecule that exerts numer- ous effects on a broad range of cell types by activating the receptor tyrosine kinase c-Kit [1]. SCF is produced by endothelial cells, fibro- blasts, keratinocytes, gut epithelial cells and tumor cells [2,3]. SCF can act on hematopoiesis by stimulating the survival and prolifer- ation of stem cells and progenitor cells [4]. It is also crucial for mast cell proliferation, function [5–7] and the development of other cells, including melanocytes and germ cells [8]. Some tumor cell proliferation and invasiveness are promoted by SCF [9]. SCF exists naturally as a membrane-bound protein composed of either 220 or 248 amino acids and soluble isoform (SCF 164 ) as a result of alterna- tive RNA splicing and proteolytic processing [9,10,11]. The soluble protein (SCF 164 ), which has two intramolecular disulfide bonds (Cys4-Cys89 and Cys43-Cys138), is a noncovalently associated dimer under non-denaturing conditions [10]. The glycosylated or unglycosylated form does not influence biological activity [7,12]. SCF in clinical trials have been performed in the treatment of ane- mia [13] and gene therapy to stimulate and expand hematopoietic stem cells (HSCs) [14]. Recombinant human SCF (rhSCF) can main- tain hematopoietic stem cells and mast cell in culture [15,16], and it is widely tested in research into differentiation from embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC) [17,18]. http://dx.doi.org/10.1016/j.pep.2014.09.015 1046-5928/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding authors at: Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan (T. Akuta). E-mail addresses: t.akuta@kyokutoseiyaku.co.jp (T. Akuta), takane@med. teikyo-u.ac.jp (T. Kikuchi-Ueda). Protein Expression and Purification 105 (2015) 1–7 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep