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.

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.

Effects of interleukin-11 on carboplatin-induced thrombocytopenia in rats and in combination with stem cell factor.

The effects of interleukin-11 (IL-11) which stimulates megakaryopoiesis and thrombopoiesis and/or stem cell factor (SCF) which stimulates multiple lineage cells in the peripheral blood by an increase in marrow cellularity on hematopoietic suppression in carboplatin (CBDCA)-treated rats were determined. CBDCA was administered to rats at a dose of 35 mg/kg as a single intraperitoneal bolus on day 0. IL-11 (20 micrograms/day) was given subcutaneously for 10 days from day 5 after CBDCA treatment. IL-11 ameliorated the suppression of hematopoiesis in rats treated with CBDCA. Especially, the production of the platelets was stimulated by IL-11 more markedly than that of cells of other lineages. However, the combination of SCF and IL-11 intensified the CBDCA-induced suppression of hematopoiesis. These findings may be helpful in the design of future therapies to prevent or treat thrombocytopenia induced by chemotherapy.