胆汁三烯结合蛋白在大肠杆菌中的表达、复性与纯化  

Expression,Refolding and Purification of Bilin Binding Protein in Escherichia coli

作　　者：李晓芳[1] 宁保安[2] 胡红芳[2] 杨丽丽[1] 左佳[1] 王彩红[2] 高志贤[2] 

机构地区：[1]华中农业大学食品科技学院,湖北武汉430070 [2]军事医学科学院卫生学环境医学研究所,天津300050

出　　处：《生物技术通讯》2009年第2期191-194,共4页Letters in Biotechnology

基　　金：国家科技支撑计划(2006BAK02A09);天津市应用基础及前沿技术研究计划重点项目(09JCZDJC17700)

摘　　要：目的:合成胆汁三烯结合蛋白(BBP)基因并在大肠杆菌中表达,获得重组BBP纯化制品。方法:根据天然BBP的基因序列和大肠杆菌偏好密码子设计并合成BBP基因的引物,PCR扩增优化的BBP基因序列,克隆至载体pEasy-T3;测序正确后,将该序列克隆至表达载体pET-32a上,构建表达质粒,转化至大肠杆菌BL21(DE3)pLysS,在IPTG诱导下表达融合蛋白;采用Ni柱纯化融合蛋白。结果:PCR扩增获得了优化后的BBP基因序列,构建了表达载体pET-32a-BBP;SDS-PAGE分析表明表达的融合蛋白相对分子质量为20×103,以包涵体形式存在,占全菌蛋白的40%以上;变性、复性后经Ni2+柱纯化,获得纯度达98%以上的重组蛋白。结论:优化并合成了BBP全基因序列,获得了高纯度重组融合蛋白,为进一步鉴定其生物活性及筛选小分子的研究奠定了基础。Objective： To synthesize bilin binding protein（BBP） gene sequence, express BBP efficiently in Escherichia coli and purify the recombinant protein. Methods： The primers were designed according to the natural BBP gene sequence and E.coli codon preference. The optimized sequence was amplified by PCR and was inserted into pEasy-T3. After determined by sequence analysis, the BBP gene was cloned into plasmid pET-32a to construct the expression vector pET-32a- BBP. The recombinant protein was over-expressed in E.coli BL21（DE3）pLysS and purified by immobilized metal ion affinity chromatography. Results： The BBP gene sequence was obtained by PCR. The expression plasmid named pET-32a-BBP was constructed and the recombinant protein was expressed in E.coli BL21 （DE3）pLysS as inclusion body, accounting for 40% of the total bacterial proteins. After denaturation and refolding, the fusion protein was successfully purified by Ni^2＋ affinity chromatography. Conclusion： Purified recombinant BBP was obtained, determination of its biological activity and screening of small molecule binding protein are the next steps.

关 键 词：胆汁三烯结合蛋白 大肠杆菌 融合蛋白 

分 类 号：Q78[生物学—分子生物学]