[Cloning, expression and purification of human stem cell growth factor cDNA and its species-specificity in hematopoiesis].
Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.
Yuan
Ye
Y
Department of Cell Biology, Beijing Institute of Basic Medical Sciences, Beijing 100850, China.
Zhang
Yun-Sheng
YS
Li
Xiou-Sen
XS
Guo
Zi-Kuan
ZK
Liu
Xiao-Dan
XD
Hou
Chun-Mei
CM
Tang
Pei-Xian
PX
Mao
Ning
N
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D013045:Species Specificity; D019089:Stem Cell Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D019089:Stem Cell Factor