甜蛋白大肠杆菌工程菌株发酵条件的优化及重组蛋白纯化方法的研究  

Research of Fermentation Condition and Purification of Monellin in the Recombinant E.coli Stain

作　　者：陈忠军[1] 蔡恒[2] 路福平[3] 李正英[4] 杨志华[1] 

机构地区：[1]内蒙古农业大学食品科学与工程学院,内蒙古呼和浩特010018 [2]南京工业大学制药与生命科学学院,江苏南京210009 [3]天津科技大学生物工程学院,天津300222 [4]内蒙古农业大学职业技术学院,内蒙古包头014109

出　　处：《华北农学报》2006年第F12期22-26,共5页Acta Agriculturae Boreali-Sinica

基　　金：内蒙古自然科学基金(NO2006070105190)

摘　　要：将带有monellin基因的重组分泌型表达载体pETMO转入大肠杆菌BL21(DE3)。得到重组工程菌株BLC01。研究不同的表达条件对甜蛋白表达水平的影响,得到优化发酵条件:装液量为50ml/250ml三角瓶,当培养液OD值达0.8时,添加诱导剂IPTG至浓度为0.9mM,32℃诱导5h。此时,甜蛋白monellin表达量可高达细菌可溶性蛋白的45.2%。超声破碎细胞后,利用热处理和酸处理结合的方法有效去除宿主蛋白,透析后利用Sephadex G- 50进行柱层析,得到纯化重组甜蛋白monellin。monellin基因在重组大肠杆菌中的表达产物具有生物活性。A expression vector pETMO harboring the single-chain monellin gene was transferred into E.coli BL21（DE3）.In this study, the optimal condition of fermentation was obtained. The recombinant E.coli was grown at 37℃ with shaking at 200 rpm. When OD600nm of the culture reached 0.8, IPTG was added to give a final concentration of 0.9 raM, and then incubated at 32℃ for 5 h to over-express the recombinant moneUin. Monellin was produced accounting for 45.2% of total soluble proteins. After broken up by ultrasonic the cell was treated by acid and heating to remove effectively the protein of host, the liquid was dialyzed and then sublimated by Sephadex G-50 to obtain pure monellin. The E. coli-expressed monellin has active.

关 键 词：甜蛋白MONELLIN 重组菌株 发酵条件 纯化 

分 类 号：Q786[生物学—分子生物学] TQ460.38[化学工程—制药化工]