粒细胞集落刺激因子(G-CSF)在大肠杆菌中的高效表达及快速纯化工艺的建立  

Over expression of G CSF in E.coli and Establishment of Fast Purification Protocol

作　　者：李福胜[1] 贡惠宇[1] 赵炳文 余彩玲 侯斌 陈爱君[1] 张智清[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室

出　　处：《中国生物化学与分子生物学报》1998年第5期479-484,共6页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：基因工程重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）主要用于癌症患者化疗后的粒细胞减少症．在正确克隆人Ｇ－ＣＳＦｃＤＮＡ的基础上，重点对Ｇ－ＣＳＦｃＤＮＡ的５′端进行了较为彻底的修饰，修饰后的基因插入ｐＢＶ２２０载体组建成功ｐＢＶ２２０／Ｇ－ＣＳＦ／２－１７４高效表达载体．表达后ＳＤＳ－ＰＡＧＥ分析其表达最高达５０％以上．根据Ｇ－ＣＳＦ表达形成包涵体这一特性，建立了一条简便、稳定，适用于大规模生产的分离纯化工艺流程．首先分离纯化包涵体，８ｍｏｌ／Ｌ尿素裂解包涵体，稀释复性蛋白，之后一步ＳＰ－ＳｅｐｈａｒｏｓｅＦＦ柱层析至均质．纯化的Ｇ－ＣＳＦ比活性达３．４×１０８Ｕ／ｍｇ蛋白，每升表达菌液回收的Ｇ－ＣＳＦ总活性达１．０６×１０１１Ｕ．纯化产物的Ｎ－端氨基酸序列分析表明，对甲硫氨酸的去除彻底。Recombinant human granulocyte colony stimulating factor (rhG CSF) is mainly used in neutropenia induced by cytotoxic chemotherapy in clinical practice.After Chinese human G CSF cDNA was cloned,the 5′ terminal sequence in G CSF cDNA was thoroughly modified in order to raise the expression level.pBV220/G CSF/2 174 was constructed by inserting the modified gene into pBV220 vector.At last,over 50% expression level was achieved as analyzed by SDS PAGE.As G CSF was in inclusion body in E.coli ,a simple and stable purification protocol was established,which was very suitable for large scale purification.Firstly,inclusion body was extracted from E.coli ,and then,8 mol/L urea was used to lysis the inclusion body.G CSF protein was renatured by dilution.At last,the pure G CSF was recovered by one step SP Sepharose FF chromatography.The relative activity of purified G CSF reached to 3 4×10 8 U/mg protein.A total G CSF activity from 1 L fermentation was about 1 06×10 11 U.As demonstrated by N terminal amino acid sequencing,methionine had thoroughly been removed,so this kind of purified G CSF may have a low immunogenicity and toxicity.

关 键 词：G-CSF 原核表达 纯化 PBV220 基因修饰 

分 类 号：Q78[生物学—分子生物学]