牙龈卟啉单胞菌pPGN蛋白的原核表达、纯化及生物信息学分析

Prokaryotic expression,purification and bioinformatics analysis of pPGN protein in Porphyromonas g gival

目的 孢子相关重复(Sporulation-related repeat, SPOR)结构域作为一种小肽聚糖结合结构域,在细胞分裂过程中起着重要作用。来自牙龈卟啉单胞菌的pPGN蛋白含有SPOR结构域,但其结构和功能尚不清楚。本研究原核表达、纯化pPGN蛋白,并进行生物信息学分析,为新型药物靶点的研发提供参考。方法 构建pET-28a(+)-pPGN表达载体,以大肠埃希菌为宿主菌异源诱导表达目的蛋白,经Ni-NTA亲和层析及分子筛纯化后使用ITC进行功能初探,采用生物信息学方法预测分析其理化性质与结构功能。结果 1%琼脂糖凝胶电泳分析显示,重组表达载体构建成功;SDS-PAGE电泳显示纯化得到较高纯度的pPGN蛋白单体;ITC显示pPGN蛋白与N-乙酰氨基葡萄糖(NAG)、N-乙酰胞壁酸(NAM)之间不存在相互作用关系。生物信息学分析pPGN蛋白为碱性稳定性蛋白,无跨膜区域,含有24个磷酸化位点和一个保守结构域(SPOR结构域)。SPOR结构域蛋白比对分析显示,pPGN蛋白与RlpA(6i09-A)和CwlC(1x60-A)蛋白的三级结构高度相似。分子对接分析显示pPGN蛋白与三糖(NAM-NAG-NAM)相互作用的残基主要为R100、R105、R139,且作用于两端的NAM,与pPGN蛋白相互作用的蛋白有10种。结论 pET-28a(+)-pPGN表达载体在原核系统(大肠埃希菌)中得到高效表达,纯化获得具有高纯度的pPGN蛋白。分子对接分析其含有与NAM-NAG-NAM相结合的作用位点残基,为该蛋白的结构与功能研究奠定了基础,也为新型药物靶点的研发提供了一条新思路。

Objective The Sporulation-related repeat(SPOR)domain plays an important role in cell division as a small peptidoglycan binding domain.The pPGN protein from Porphyromonas gingivalis contains the SPOR domain,but its structure and function are unknown.In this study,prokaryotic expression and purification of pPGN protein were carried out and bioinformatics analysis was carried out to provide a reference for the development of new drug targets.Methods The expression vector of pET-28a(+)-pPGN was constructed,with Escherichia coli as the host bacterium heterologous induced expression of the target protein.After Ni-NTA affinity chromatography and molecular sieve purification,ITC was used to conduct preliminary functional exploration.Finally,bioinformatics methods are used to predict and analyze its physicochemical properties and structural functions.Results The electrophoresis analysis of 1%agarose gel showed that the recombinant expression vector was successfully constructed.SDS-PAGE electrophoresis showed that pPGN protein monomers of higher purity were obtained after purification by affinity chromatography and molecular sieve.ITC showed that there was no interaction between pPGN protein and N-acetylglucosamine(NAG)and N-acetylmuramic acid(NAM).Bioinformatics analysis pPGN protein is a basic stable protein with no transmembrane region,containing 24 phosphorylation sites and a conserved domain(SPOR domain).SPOR domain protein alignment analysis showed that the pPGN protein was highly similar to the tertiary structure of RlpA(6io9-A)and CwlC(1x60-A)proteins.Molecular docking analysis showed that the residues of pPGN protein interacting with triosaccharides(NAM-NAG-NAM)were mainly R100,R105,R139,and acted on NAM at both ends,and there were 10 proteins interacting with pPGN protein.Conclusion The pET-28a(+)-pPGN expression vector is efficiently expressed in the prokaryotic system(Escherichia coli),and the pPGN protein with high purity is purified.Molecular docking analysis contains site residues bound to NAM-NAGNAM,which lays the foundation for the study of the structure and function of the protein,and also provides a new idea for the development of new drug targets.