Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor.

Sangki Baek,Yeon Jin Jang,Michelle Novais de Paula,Chong Jai Kim,Minh Tan Nguyen,Huynh Kim Khanh Ta,Han Choe,Eunyoung Lee,Taeck-Hyun Jeong

doi:10.3390/ijms22126361

Abstract

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Highlights

Human stem-cell factor, known as the c-Kit ligand, mast cell growth factor, or steel factor, stimulates hematopoiesis, stem-cell maintenance, gametogenesis, mast-cell development, and melanogenesis [1,2,3,4,5,6]. hSCF has been used for the differentiation of stem cells into a range of specialized cells, such as endothelial cells, erythropoietic cells, lymphoid progenitors, osteoclasts, and spermatozoa [7]
Three constructs and one construct for expression in E. coli were designed for hSCF164 and hSCF248, respectively (Figure 1b)
The tobacco etch virus protease restriction site (TEVrs), ENLYFQG, was placed between the tag and hSCFs to remove the tag during the purification

Summary

Introduction

Human stem-cell factor (hSCF), known as the c-Kit ligand, mast cell growth factor, or steel factor, stimulates hematopoiesis, stem-cell maintenance, gametogenesis, mast-cell development, and melanogenesis [1,2,3,4,5,6]. hSCF has been used for the differentiation of stem cells into a range of specialized cells, such as endothelial cells, erythropoietic cells, lymphoid progenitors, osteoclasts, and spermatozoa [7]. Human stem-cell factor (hSCF), known as the c-Kit ligand, mast cell growth factor, or steel factor, stimulates hematopoiesis, stem-cell maintenance, gametogenesis, mast-cell development, and melanogenesis [1,2,3,4,5,6]. HSCF has been used for the differentiation of stem cells into a range of specialized cells, such as endothelial cells, erythropoietic cells, lymphoid progenitors, osteoclasts, and spermatozoa [7]. Primordial germ cells have been reported to be induced from human pluripotent stem cells by using hSCF. HSCF has been used in the generation of humanized mice [8]. HSCF has assisted in the recovery of cardiac function following myocardial infarction by increasing the number of cardiomyocytes and vascular channels [9].

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