重组人白血病抑制因子在大肠杆菌中的表达及纯化  

Expression and Purification of Recombinant Human Leukemia Inhibitory Factor from E.coli

作　　者：王晓雯[1] 郗雪艳[1] 李小璐[2] 关欣[2] 郭阳[1] 宋伟[2] 杜伯雨[1] 卫荣华[1] WANG Xiao-wen XI Xue-yan LI Xiao-lu GUAN Xin GUO Yang SONG Wei DU Bo-yu WEI Rong-hua(School of Basic Medicine, Hubei University of Medicine, Shiyan, Hubei 442000 Department of Biochemistry, Institute of Basic Medi- cine, Chinese Academy of Medical Sciences ＆ Peking Union Medical College,Beijing 100005, China)

机构地区：[1]湖北医药学院基础医学院,湖北十堰442000 [2]北京协和医学院基础学院中国医学科学院基础医学研究所生化学系,北京100005

出　　处：《湖北医药学院学报》2017年第3期214-220,共7页Journal of Hubei University of Medicine

摘　　要：目的:利用大肠杆菌表达并制备重组人源白血病抑制因子(Leukemia inhibitory factor,LIF)。方法:构建p ET-30a-rh LIF表达载体并转入大肠杆菌菌株BL21(DE3)之中,IPTG诱导使无标签的目的蛋白呈可溶性表达,继而利用三步柱层析纯化目的蛋白。最后对所获目的蛋白活性进行测定,并对其N端和C端的完整性分别应用Edman测序法以及质谱法进行鉴定。结果:通过三步柱层析以及中间的内毒素去除步骤可使无标签rh LIF获得纯化,检测结果提示所获目的蛋白内毒素水平<1 EU/μg,其活性同市售商品化LIF产品一致,Edman测序结果提示少部分目的蛋白N端存在甲硫氨酸修饰,质谱检测结果表明所获目的蛋白C端完整性较好。结论:建立了一种新的重组人源白血病抑制因子表达和纯化的方法,本方法不需利用标签蛋白,并可有效降低所获蛋白中内毒素水平,所获rh LIF蛋白可用于干细胞培养以及相关生物学研究。Objective To prepare recombinant human leukemia inhibitory factor（ rh LIF） from E. coli BL21. Methods The expression vector p ET-30a-rh LIF was constructed and transformed into E. coli BL21（ DE3）. After the soluble expression of untagged rh LIF was induced after IPTG addition,the three-step chromatography was employed for the purification of rhLIF protein. The activity of purified LIF was determined and compared with that of the commercial product. The integrity of N terminal and C terminal amino acid sequences of the purified rh LIF protein was verified by Edman degradation assay and Mass spectrometry analysis. Results The untagged rh LIF protein could be purified through three-step chromatography and an intermediate endotoxin removal step. The endotoxin level of purified rh LIF protein was below 1 EU/μg,which was comparable to that of the commercial product. The N-terminal integrity of purified rh LIF protein was confirmed by Edman degradation assay,and methionine modification at N-terminal amino acid was identified in a small number of target proteins.The integrity of C-terminus of rh LIF was also confirmed with mass spectrometry. Conclusion A novel method for expression and purification of recombinant human leukemia inhibitory factor had been established. In this method,no affinity tags were involved thereby facilitating the purification process. Meanwhile,the endotoxin level could be effectively reduced to an acceptable level. It had been proved that the purified rh LIF by this method could be used for stem cell culture and other related biological studies.

关 键 词：人白血病抑制因子 表达 纯化 大肠杆菌 

分 类 号：R346[医药卫生—基础医学]