摘要
为了提高重组人胰岛素的表达量,用E.coli发酵表达(His)6-Arg-Arg-人胰岛素原[(His)6-Arg-Arg-human proinsulin,RRhPI],并对影响RRhPI高效表达的各主要因素以及下游纯化工艺的各关键步骤进行优化。研究表明,RRhPIE.coli在最优培养基:5 g/L胰蛋白胨,2 g/L谷氨酸,7 g/L酵母浸膏,0.5 g/L酸铵,2.5 g/L葡萄糖,2.7 g/L甘油,100 mg/L氨苄青霉素,50 mg/L卡那霉素,pH7.2的条件下,用发酵罐进行发酵,在对数生长中期加入0.5 mmol/L IPTG诱导4 h,发酵过程中通过补料和转速调节,每升培养基能收获湿菌体51.2 g,包涵体16.2g。在下游纯化工艺中,初步纯化前包涵体溶解液中一定浓度的β-巯基乙醇以及复性液中一定浓度的氧化型谷胱甘肽将有助于RRhPI的复性。经过工艺条件的优化,人胰岛素最终收率可达74 mg/L培养基。
To obtain recombinant human insulin （ rhI）, （His） 6 - Arg - Arg - human proinsulin （RRhPI） was expressed by Escherichia coli（E, coli）. In the optimum medium of 5 g/L tryptone,2 g/L glutamic acid,7 g/L yeast extract ,0.5 g/L ammonium sulfate ,2, 5 g/L glucose ,2.7 g/L glycerol, 100 mg/L ampicillin ,50 mg/L kanamycin, and pH 7.2, and with the addition of 0.5 mmol/L IPTG at intermediate exponential phase and induction for 4 h , 51.2 g wet E. coli and 16.2 g inclusion body were harvested from 1 L medium in fermenter. The results showed that certain concentration of β - mercaptoethanol in inclusion body solution and oxidized glutathione in refolding solution were helpful to RRhPI refolding. With the well-optimized technology, 74 mg rhI was obtained from 1 L culture medium.
出处
《四川大学学报（工程科学版）》
EI
CAS
CSCD
北大核心
2007年第4期79-83,共5页
Journal of Sichuan University (Engineering Science Edition)
基金
科技部创新基金资助项目(06C26215100476)
