大肠杆菌表达的重组猪β_2微球蛋白二级结构的圆二色谱分析

Analyzing secondary structure of β_2 microglobulin in Escherichia coli with circular dichroism spectrum

【目的】研究巴马小型猪β2微球蛋白(β2m)的结构和功能。【方法】亚克隆巴马小型猪β2m的成熟肽部分,并构建与pMAL-p2X的重组质粒,转化大肠杆菌TB1并诱导后,融合表达蛋白分别经过Western blot、纯化及Factor Xa切割,分离纯化单体蛋白。圆二色谱(circular dichroism spectrum,CD)测定蛋白的二级结构。【结果】重组表达的融合蛋白MBP-β2m大小为53.1kDa。切割后去除MBP的单体蛋白大小为10.6kDa。圆二色谱分析单体蛋白β2m二级结构元件α-螺旋、β-折叠、转角和随机卷曲所含有的氨基酸残基数目分别为0、45、8和45。【结论】分析揭示表达的β2m具有正确的二级结构,可以用于体外多肽结合等研究。

[ Objective] In order to study the structure and function of β2 microglobulin （β2m）. [ Methods] We sub-cloned the mature peptide of β2m into the p2X plasmid and transformed them to Escherichia coli TB1. The recombinant bacterium was induced to be expressed and the expressed fusion protein was detected by SDS-PAGE and western blot. After purifying and cleaving with Factor Xa, we separated the monomer protein β2m from MBP. Finally, we determined the secondary structure of the β2m protein by circular dichroism （CD） spectrum. [Results] MBP-β2m was 52.1 kDa, and the monomer protein β2m was 10.6 kDa. The α-helix, β-sheet, turn, and random coil of the fusion protein showed 0, 45, 8 and 45 amino acids, respectively, detected by CD estimation. [ Conclusion] The β2m protein had correct secondary structure and could be used for further research of peptide binding in vitro.