整合素αMβ2 I-结构域的基因合成和蛋白表达

Gene Synthesis and Protein Expression of Integrin αMβ2 I-Domain

目的:利用大肠杆菌表达系统表达整合素αMβ2 I-结构域,并对其进行纯化。方法与结果:利用DNAWorks在线引物程序设计经过密码子优化的整合素αMβ2 I-结构域的寡核苷酸序列;采用PCR介导的基因拼装法合成αMβ2 I-结构域DNA模板,将其克隆至原核表达载体pET11d-6h上,并转化大肠杆菌,获得表达菌株;经IPTG诱导,αMβ2 I-结构域在大肠杆菌BL21(DE3)中获得高效表达,表达产物经Ni-NTA琼脂糖层析柱纯化后,纯度达到90%以上;电喷雾电离质谱测定表明纯化所得的蛋白精确的相对分子质量为23720.0,与预期值相符;肽指纹质谱图谱鉴定其为目的蛋白。结论:αMβ2 I-结构域的高效表达,为进一步研究其与配体的结合及其复合物晶体结构奠定了基础。

Objective： To express and purify αMβ2 I-domain in E.coli BL21（DE3）. Methods and Results： The cDNA encoding αMβ2 I-domain was determined according to human αMβ2 I-domain sequence protein and the E.coli bias codon. Then, it was assembled by PCR using a series of primers designed by DNAWorks, and it was cloned into pET11d-6h plasmid. The recombinant protein was expressed highly in E.coli BL21（DE3）, its purity reached 90% after it was purified by a single step affinity chromatography with Ni-NTA. Its molecular weight was the expected 23 720.0 Da measured by ESI-MS. It was characterized by peptide mass fingerprinting and identified to be the recombinant αMβ2 I- domain. Conclusion： Highly purified αMβ2 I-domain provides a basis for further structure studies of αMβ2 I-domain-lig-and complexes.