Expression and characterization of human G-CSF by E. coli

• Main Authors: Kai-long Wu, 巫凱隆
• Other Authors: Kee-jong Chang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/26464112299275278855

碩士 === 東吳大學 === 化學系 === 94 === Granulocyte colony-stimulating factor (G-CSF) is an essential cytokine in the hemotopoietic process that stimulates the proliferation and differentiation of neutrophils specifically. Nowadays, recombinant G-CSF is widely used for the treatment of neutropenia caused by various origins as well as in bone marrow transplantation. It is one of the most successful recombinant proteins on the market so far. In this study, the cDNA of human G-CSF with a 6 His-tag at 5' end was synthesized by PCR and inserted into the pET25b expression vector under the control of T7 promoter then transformed into E. coli strain BL21 (DE3). After 3 hr induction by IPTG at 37℃, the high level expression of the recombinant G-CSF was achieved and confirmed by western blotting where it represented approximately 80% of the total cellular protein as determined by SDS-PAGE. The amount of recombinant G-CSF was 100 mg in 500 ml LB medium approximately. The recombinant G-CSF was purified under the denatured conditions and followed by the enterokinase digestion to obtain the authentic cytokine.