摘要
旨在优化重组人抗血栓蛋白(rHAP)工程菌的自诱导培养基,以提高菌体产量及可溶性的重组rHAP蛋白含量。选取蛋白胨、酵母提取物、甘油、葡萄糖为因素,各取4个水平,采用正交表L16(45)进行试验设计,对影响菌体生长和可溶性rHAP表达水平的乳糖浓度进行了优化。结果表明自诱导培养基碳氮源的最优配比:2%蛋白胨,1.5%酵母,0.5%甘油,0.03%葡萄糖,0.2%乳糖。此时表达菌密度OD600和可溶性rHAP目标蛋白表达量分别是未优化前的2.04倍和2.85倍。在20 L发酵表达时,rHAP工程菌OD600值高达93,菌体湿量为1 620 g/20 L。利用Q-Sepharose和SP-Sepharose纯化,Western blotting结果表明表达蛋白为目的融合蛋白。
In order to increase the growth and the soluble expression of recombinant E.coli strain expressing human anticoagulant protein（rHAP）,auto-induction medium were optimized.Tryptone,yeast extract,glycerol and glucose（four level for each substance and peptone used as factor and biomass and soluble expression of rHAP used as objective product respectively）were used for L16（45）orthogonal experiment,concentrations of lactose which could affect the growth of recombinant bacterial strain and product of rHAP were investigated.Result showed that the optimal carbon and nitrogen composition ratio in auto induction medium was obtained as followed： 2% tryptone,1.5% yeast extract,0.5% glycerol,0.03% glucose,0.2% lactose.The optimized cultivation could increase the bacterial growth density and the product of soluble expressed rHAP protein by 2.04 fold and 2.85 fold respectively compared with the original,non-optimized condition.When fermented in 20 Liter fermentor,the recombinant rHAP-expressing bacteria grew to OD600 of 93 with the wet weight of bacteria of 1 620 g/20 L.The recombinant protein of rHAP was highly purified by Q-Sepharose and SP-Sepharose,which was identified by Western blotting.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第12期192-198,共7页
Biotechnology Bulletin
基金
江苏省自然科学基金(BK2010046
BK2008138
BE2008639
BY2009147)
"重大新药创制"科技重大专项(2009ZX09103-675
2009ZX09102-206)
常州市科技局(CS20092003
CQ20100009
CN20100016
CZ20100008)
常州市武进区科技局资助项目
关键词
抗血栓蛋白
优化培养
发酵
纯化
鉴定
Anticoagulant protein Optimization of cultivation Fermentation Purification Identification
