Prokaryotic Expression For Chinese Soluble Stem Cell Factor Gene And Its Purification

Objective: To construct prokaryotic expression vector for soluble human stem cell factor gene (rhSCF) and to express recombinant stem cell factor in E.coli BL21. Methods: Total RNA was extracted from fresh marrow by Trizol Reagent, and human stem cell factor cDNA was amplified by RT- PCR , then cloned into vector pMD18 -T for sequencing. Prokaryotic expression vector pET32a (+ ) / SCF was constructed and expressed in E. coli BL21 with induction of IPTG. The fusion protein was purified with Ni-NTA agarose resin.Result: In this study, recombinant stem cell factor cDNA cloned was 500bp in length. DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence. SDS-PAGE analysis showed the prokaryotic expression vector pET32a( + ) / hSCF fusion protein with relative molecule mass 37KD was expressed in E. coli BL21 ,and the amount of it is about 30% of the total bacterial protein after being induced by IPTG for 3 h. The product was obtained with a purity of about 90%. Conclusion: Constructed pET32a ( + ) /hSCF is efficiently expressed in E. coli BL21.