Raf-1蛋白突变体的原核表达、纯化及活性鉴定  

Prokaryotic expression and purification of Raf-1 mutant and evaluation of its bioactivity

作　　者：刘小会[1] 高学娟[1] 刘朗夏[1] 

机构地区：[1]暨南大学生命与健康工程研究院,广州市510632

出　　处：《实用医学杂志》2012年第10期1599-1602,共4页The Journal of Practical Medicine

基　　金：国家973重点基础研究发展计划(编号:2011CB910701);广东省自然科学基金(编号:9151065004000005);暨南大学特殊人才启动基金(编号:50624001);广东省高等学校高层次人才项目(编号:50524006)

摘　　要：目的:将Raf-1蛋白259位丝氨酸(Ser,S)突变成天冬氨酸(Asp,D),构建突变型GST-Raf-1220-268S259D原核表达载体,表达和纯化融合蛋白及鉴定其生物学活性。方法:以pGEX-4T-1-Raf-1220-268为模板,运用PCR定点突变技术扩增出Raf-1220-268S259D基因序列,插入原核表达载体pGEX-6P-1,表达和纯化重组蛋白,GST沉降实验鉴定蛋白的生物学活性。结果:重组质粒测序结果正确,获得模拟磷酸化GST-Raf-1220-268蛋白。GST沉降实验检测到目的蛋白和14-3-3蛋白具有体外特异性结合活性。结论:成功构建GST-Raf-1220-268S259D原核表达载体,并表达及纯化了有生物学活性的融合蛋白,为进一步针对Raf-1的研究提供实验依据。Objective To construct and express Raf-1220-268S259D gene mutant and evaluate the bioactivity of the expressed product.Methods Raf-1220-268S259D cDNA was amplified by PCR site-directed mutagenesis with pGEX-4T-1-Raf-1220-268 as template,and then was inserted into the prokaryotic expression vector pGEX-6P-1.The recombinant vector was then transformed into the E.coli BL21（DE3） strain to produce the fusion protein,which was purified through a Sepharose-Glutathione column.The biological activity was identified by GST pull-down assay.Results The sequences of Raf-1220-268S259D were correct,and the phosphorylation of Raf-1220-268 was obtained.The fusion protein could be bound to 14-3-3 in the GST pull-down assay.Conclusions The GST-Raf-1220-268S259D plasmid was successfully constructed and the bioactive protein was obtained,which lay a foundation for further research on Raf-1 protein.

关 键 词：Raf-1蛋白 突变体 原核表达 纯化 

分 类 号：R363[医药卫生—病理学]