原核表达融合型血管生成抑制剂Kringle 5发酵条件的优化和初步纯化  

Optimization of Batch Fermentation and Preliminary Purification for Fusion Angiogenesis Inhibitor Kringle 5 in Prokaryotic System

作　　者：边六交[1] 党红艳[1] 杨晓燕[1] 

机构地区：[1]西北大学西部资源生物与现代生物技术教育部重点实验室,陕西西安710069

出　　处：《高校化学工程学报》2006年第5期775-780,共6页Journal of Chemical Engineering of Chinese Universities

基　　金：陕西省科技攻关项目(2003K10-G58)。

摘　　要：在构建融合型Kringle5原核表达重组菌BL21(DE3)pET32a/K5的基础上,通过改变培养基的成分,确定了适宜于重组菌生长和融合Kringle5蛋白表达的培养基。采用正交实验方法,对融合型血管生成抑制剂Kringle5原核表达重组菌的发酵条件进行了优化,通过发酵罐中重组菌的分批培养,确定了培养液中的最佳溶氧量。采用优化后的发酵工艺,在20L发酵罐中融合Kringle5的表达量可达0.59g.L·1。最后在Cu2+螯合的SepharoseFastFlow层析介质上对融合Kringle5进行了初步纯化,其纯度约为80%。The optimization of batch fermentation and preliminary purification of fusion angiogenesis inhibitor Kringle5 in prokaryotic system were studied. After angiogenesis inhibitor Kringle 5 and plasmid pET32a were reconstituted and transferred into E. coli BL21 （DE3）, a novel recombination E. coli BL21 （DE3） pET32a/K5 expressing fusion Kringle 5 was constructed. The optimization of fermentation culture composition for recombination E.coli BL21（DE3） pET32a/K5 growing and fusion Kringle 5 expression was investigated. The optimization of batch fermentation was studied by orthogonal experiment, the optimized fermentation conditions are： temperature 35℃, pH 7.0, the amount of inoculation 8%, agitation 260 r.min^-1, the concentration of inducer 0.8 mmol.L^-1, induced phase OD600 0.6 and induced time 6 h. By batch cultivation in fermenter, the optimal dissolved oxygen in fermentation solution was founded to be 40%. Based on the above optimal processing variables, the expression of fusion Kringle 5 can reach 0.59 g.L^-1. The fusion Kringle 5 was purified on Cu^2＋ chelated on Sepharose Fast Flow, and its purity is about 80%.

关 键 词：血管生长抑制剂Kringle 5 分批发酵 发酵条件优化 纯化 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]