一种重组人睫状神经营养因子突变体的分离纯化及结构鉴定  

Purification and Characterization of a New Recombinant Cilinary Neuronotrophic Factor Mutant Expressed in Soluble Form by E.coli

作　　者：冯翠[1] 赵大伟[2] 张纯[3] 王健[4] 秦培勇[1] 刘永东[2] 苏志国[2] 

机构地区：[1]北京化工大学生命科学与技术学院,北京100029 [2]中国科学院过程工程研究所生化工程国家重点实验室,北京100190 [3]四川大学华西药学院,成都610041 [4]北京生物制品研究所有限责任公司,北京100024

出　　处：《中国生物工程杂志》2013年第10期21-27,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(209706178)

摘　　要：研究并建立从细胞破碎上清中获得一种含游离半胱氨酸的重组人睫状神经营养因子突变体(CNTF-C17)的分离纯化方法并对纯化产物的结构及活性进行表征。通过疏水层析、离子交换和亲和层析的组合层析路线,能实现目标蛋白与其它杂蛋白的有效分离。组合层析后CNTF-C17的纯化倍数可达11.4,收率35.5%。反相高效液相色谱和凝胶过滤层析证明产物纯度可达到98%以上,且以单体形式稳定存在,无分子间二硫键结合的二聚体。质谱测得产物分子量与理论值一致,圆二色光谱和荧光光谱证明纯化产物具有正确的二、三级结构,TF-1.CN5a.1细胞增殖活性检测表明纯化产物比活达到2.1×106IU/mg。因此,为这种新的CNTF突变体规模化生产及后续应用奠定了基础。Ciliary neurotrophic factor （CNTF） can promote the survival and differentiation of motor neurons and is a potential therapeutic for treating neurodegeneration and nerve injury. Recent research finds it also has great potential as a diet reducing aid. In order to develop its site-specific modified long-acting counterpart, a new human CNTF mutant with a free cystine （CNTF-C17） expressed in soluble form by E.coli has been purified and featured. By combining three different chromatographic steps including hydrophobic interaction chromatography, ion exchange chromatography and affinity chromatography, CNTF-C17 was efficiently separated from bacterial contaminants with a purification factor of 11.4 and a recovery of 35.5%. The purity of target protein reached 98% determined by RP-HPLC. Although this new mutant has a free cysteine, it was found to exist only in monomer form, judged from the elution volume in HP-SEC. CD and FL spectra showed correct secondary and tertiary structure of the purified protein. The molecular weight of this new mutant was 21146 Da, consistent with its theoretical value. Its specific bioactivity was 2.1 ×106 U/mg determined by Tf-1 cell activity detection method. All these data and results laid a foundation for the large production and application of this new CNTF mutant.

关 键 词：重组人睫状神经营养因子 半胱氨酸 可溶表达 分离纯化 组合层析 

分 类 号：Q503[生物学—生物化学]