Liu J.,Beijing Abzymo Biosciences Co. |
Chen D.,Beijing Abzymo Biosciences Co. |
Li L.,Beijing Abzymo Biosciences Co. |
Liu X.-Y.,Beijing Abzymo Biosciences Co. |
And 6 more authors.
Chinese Journal of Biologicals | Year: 2012
Objective: To synthesize Macaca mulatta granulocyte-macrophage colony stimulating factor (mGM-CSF) gene, highly express in E. coli and purify the expressed product. Methods: According to the E. coli-preferred codon, mGM-CSF gene was designed and synthesized, and cloned into prokaryotic expression vector pET-43.1a (+). The constructed recombinant plasmid pET-43.1a-mGM-CSF was transformed to E. coli BL21-CodonPlus (DE3)-RIPL and induced with IPTG. The expressed recombinant mGM-CSF was purified by Sephacryl S-200 molecular sieve chromatography, re-naturalized, then determined for reactogenicity by Western blot, and for biological activity by MTT method. Results: Both restriction analysis and sequencing proved that recombinant plasmid pET-43.1a-mGM-CSF was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 15 000, contained about 30% of total somatic protein and mainly existed in a form of inclusion body. The protein reached a purity of more than 95% after purification and re-naturalization, and showed specific binding to rat anti-human GM-CSF monoclonal antibody. The specific activity of the recombinant protein was 1.2 × 107 IU/mg. Conclusion: Recombinant mGM-CSF was successfully expressed in E. coli, which showed high biological activity after purification and re-naturalization. Source