Fermentation And Purification Of The Fusion Protein Of HSA-GCSF~m Expressed By Pichia Pastoris

Granulocyte Colony Stimulating Factor(G-CSF) is a cytokine which is produced by monocytes and fibroblasts. G-CSF is mainly used for aplastic anemia and neutropenia caused by chemotherapy and radiation. In order to prolong the half-life and improve biological activity, the plasmid pPIC9K-HSA-GCSF~m which harbored the encoding gene of mutant of human granulocyte colony stimulating factor and human serum albumin(HSA-GCSF~m)fusion protein was constructed and HSA-GCSF~mwas expressed in Pichia pastoris GS115.Based on above results, the fermentation methods of this recombinant Pichia pastoris in shake flask and fermentor have been researched. The purification methods and the biological activity of HSA-GCSF~m also have been discussed in this paper.The medium of flask fermentation for producing HSA-GCSF~mby Pichia pastoris have been investigated. By single factor experiments and response surface methodology tests, the optimal medium formula of growth phase medium by flask fermentation is obtained as follows: yeast extract 46.0 g·L-1, glycerol 41.0 g·L-1 and 100 mmol·L-1 potassium phosphate buffer(pH 6.0). Seeds liquid is inoculated to optimal growth phase medium with 3%inoculation concentration and we will obtain a cell desity of 18.16 g-DCW·L-1 in 28 hours’ time. The medium of induction phase by flask fermentation have been optimized by using single factor and orthogonal tests, and the optimal medium formula of induction phase by flask fermentation is as follows: yeast extract 10 g·L-1, peptone 30 g·L-1, methanol 2.5%, 100mmol·L-1 potassium phosphate buffer(pH 6.4). By using optimized induction phase medium,the expression level of HSA-GCSF~m will reach to 257 mg·L-1 in 84 hours’ time after methanol induction.The effects of methanol feeding control, adding tryptone and methanol/sorbitol co-feeding induction on the expression of HSA-GCSF~m in Pichia pastoris GS115/HSA-GCSF~m have been discussed in 7 L fermentor experiment. The results show that the expression of HSA-GCSF~m is only 37 mg·L-1 with supply of methanol sufficient, but expressed HSA-GCSF~mwill increase to 239 mg·L-1if methanol is supplied restrictedly.Adding tryptone with methanol supplied restrictedly can increase expression level of HSA-GCSF~m to 266 mg·L-1; and feeding sorbitol as auxiliary carbon source simultaneously,the expression level of HSA-GCSF~m will increase to 424 mg·L-1. After analyzing OUR,extracellular protease activity and carbon distribution, we conclude that restricting methanol concentration at low level and adding tryptone and sorbitol properly will improve metabolic activity of cells and reduce activity of extracellular protease.The purification method of HSA-GCSF~m has been established. Supernatant is separated by Blue Sepharose Fast Flow, Phenyl Sepharose Fast Flow and SP Sepharose Fast Flow chromatographies. The total recovery of HSA-GCSF~m is 13.1%. The purified product shows a single band in SDS-PAGE analysis and its purity is identified as 95% by HPLC. NFS-60cell/MTT colour metric test shows that the special activity of HSA-GCSF~m is 2.7×107IU·mg-1.