Zhang W.-M.,Zhejiang Uniongen Biopharm. Co. |
Hong J.,Zhejiang Uniongen Biopharm. Co. |
Lin D.-H.,Zhejiang Uniongen Biopharm. Co. |
Sun T.-W.,Zhejiang Uniongen Biopharm. Co. |
And 2 more authors.
Chinese Journal of Biologicals | Year: 2012
Objective: To develop a method for isolation and purification of long chain Arg3 human insulin-like growth factor-1 (LR3IGF-1). Methods: Recombinant Pichia pastoris with LR3IGF-1 gene was inoculated to a 30 L fermenter for high density fermentation. The fermentation liquid was concentrated by centrifugation and ultrafiltration, purified by SP Sepharose FF cation exchange chromatography, Phenyl Sepharose FF hydrophobic chromatography, S-100 gel filtration chromatography and DEAE Sepharose FF chromatography, then determined for protein concentration, based on which the recovery rate of protein was calculated, and the concentration of purified sample was analyzed. The activities of samples at various steps of purification in promoting the proliferation of HIN-3T3 cells were determined by MTT method. Results: The total recovery rate and RP-HPLC purity of purified LR3IGF-1 was 28% and more than 95% respectively. The activity of LR3IGF-1 increased with the increasing purity during purification, which was basically uninfluenced by the purification steps. Conclusion: A simple and effective procedure for isolation and purification of LR3IGF-1 was preliminarily developed, which laid a foundation of large-scale production of LR3IGF-1.