摘要
目的:通过对人Id2基因的稀有密码子进行突变并构建原核表达重组质粒,在大肠杆菌(escherichia coli,E.coli)中表达Id2蛋白,分析稀有密码子对Id2基因表达的影响。方法:PCR扩增突变Id2基因序列,然后转入E.coli BL21中进行表达,利用镍离子亲和层析、离子交换层析及分子筛纯化。结果:正确构建了Id2基因的基因突变表达质粒,基因突变表达的Id2蛋白在E.coli BL21中为可溶性上清表达,成功纯化了目的蛋白,且分子筛结果显示其在溶液中呈现二聚体状态。结论:稀有密码子对Id2蛋白在E.coli BL21中大量可溶性上清表达有重要影响。
Objective：To analyze the effects of rare codon on functional expression of inhibitor of DNA binding and/or differentiation 2（Id2） gene by mutating rare codon of human Id2 protein,constructing expressing recombinant plasmids and expressing Id2 protein with solution in escherichia coli（E.coli） system.Methods：Id2 human gene was amplified by PCR,then Id2 was expressed in E.coli BL21.Plasmids were constructed by mutating rare codes into normal ones.Expressed production was purified by nickel column chromatography,ion-exchange column chromatography and size exclusion chromatography.Results：Expression vector of Id2 was successfully constructed and the expressed aim protein in E.coli BL21 was in a soluble supernatant way.Human recombinant protein was purified and exhibited as homodimers in solutions by the size exclusion chromatography experiments.Conclusion：Rare codons are believed to play a key role in disturbing the soluble supernatant expression of human Id2 protein in E.coli.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2013年第4期365-369,共5页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:30970563)
关键词
ID2
突变
表达
纯化
Id2
mutation
expression
purification
