单增李斯特菌TatD-like DNase序列结构特征及其表达纯化和重组蛋白活性的研究

Structural characterization,expression purification and activity of recombinant protein of Listeria monocytogenes TatD-like DNase

本研究旨在探究单增李斯特菌10403s TatD-like DNase蛋白的序列结构特征与原核表达重组蛋白的核酸酶活性。应用生物信息学软件预测分析蛋白序列结构特征,构建重组原核表达质粒pET-32a-TatDlike DNase,转化入大肠杆菌Rosetta。重组蛋白经IPTG诱导表达、亲和层析纯化、透析复性后进行核酸酶活性分析。结果显示,单增李斯特菌10403s TatD-like DNase由265个氨基酸组成,理论蛋白分子质量为30.16 ku,与芽孢杆菌属TatD蛋白同源性最高。二级结构主要由49.43%的α-螺旋、29.81%的无规则卷曲和16.6%的β-折叠构成。SDS-PAGE和Western-blot显示,重组原核表达蛋白大小约52 ku,主要以包涵体形式表达。琼脂糖凝胶电泳证实,重组蛋白在Mg2+存在时切割λDNA的核酸酶活性显著增强,且最适反应温度和pH值分别为37℃和pH6.0。本研究系统性分析了单增李斯特菌TatD-like DNase的序列结构特征,得到了重组原核表达蛋白,且该重组蛋白具有Mg2+依赖性核酸酶活性,这为进一步研究TatD-like DNase在单增李斯特菌感染中的作用奠定了基础。

The aim of the present study was to investigate the sequence structure of the Tat D-like DNase protein of Listeria monocytogenes 10403 s and the nuclease activity of a recombinant protein expressed in a prokaryotic expression system.In the present study,the bioinformatics software tools were used to analyze the structural features of the amino-acid sequence,and the recombinant prokaryotic expression plasmid(pET32 a-Tat D-like DNase) was constructed,and transformed into E.coli Rosetta.The recombinant protein was expressed after IPTG induction and then purified by affinity chromatography.The nuclease activity analysis of the recombinant protein was performed after purification and renaturation.The results showed that the Tat D-like DNase of Listeria monocytogenes 10403 s was consisted of 265 amino acids,and the theoretical protein molecular weight was 30.16 ku,which was the highest homology with Tat D protein of Bacillus.The secondary structure was mainly constituted by the 49.43%α-helix,29.81%random coil and 16.6%β-sheet.SDS-PAGE and Western-blot analysis showed that the recombinant protein was about 52 ku,which was mainly expressed in the form of inclusion bodies.Agarose gel electrophoresis confirmed that the degradation activity of λDNA of the recombinant protein was significantly enhanced in the presence of Mg2+,and the optimum reaction temperature and p H were 37 ℃and p H6.0,respectively.In the present study,the sequence structure of Listeria monocytogenes Tat D-like DNase was systematically analyzed,and the recombinant prokaryotic expression protein has a Mg2+-dependent nuclease activity.This study laid a foundation for further study on the role of Tat D-like DNase in Listeria monocytogenes infection.