牙龈卟啉单胞菌ATCC 33277菌株外切聚磷酸酶的表达、纯化和结晶

外切聚磷酸酶(exopolyphosphatase,PPX)对于细菌在恶劣环境下引起的严紧反应、病原菌的致病性和抗生素耐药性等生物学过程必不可少。牙龈卟啉单胞菌是与牙周炎关系最密切的病原菌。为解析牙龈卟啉单胞菌(Porphyromonas gingivalis)ATCC 33277菌株的外切聚磷酸酶(PgPPX)的结构,首先利用大肠杆菌对PgPPX蛋白基因进行克隆与蛋白表达,然后依靠谷胱甘肽巯基转移酶标签对融合蛋白亲和层析后,采用阴离子交换色谱、凝胶过滤色谱得到表面电荷、聚合形态均一的PgPPX蛋白,最后对纯化后的目的蛋白进行结晶条件筛选。结果表明:PgPPX蛋白在溶液中表现为二聚体形式,纯度大于98%,表达量高达30 mg/L,且该蛋白在0.2 mol/L MgSO_4·6 H_2O、20%PEG3350(m/V)条件下获得的晶体形状较好。

Expression, Purification and Crystallization of the Exopolyphosphatase from Porphyromonas gingivalis ATCC 33277 Strain

Exopolyphosphatase (PPX) is indispensable for many biological processes, such as stringent response initiated by unfavorable growth conditions, pathogenesis of pathogenic bacteria and antibiotics resistance. Porphyromonas gingivalis is the most closely related pathogen of periodontitis. In order to solve the structure of exopolyphosphatase (PgPPX) from Porphyromonas gingivalis strain ATCC 33277, the gene was cloned and expressed in E. coli. The fusion protein was purified by affinity chromatography via the glutathione thiol transferase tag. Then the PgPPX protein with uniform surface charge and morphology was obtained by anion exchange chromatography and gel filtration chromatography successively. The crystallization conditions were screened further. The results suggest that PgPPX exists as a dimer in solution and the purity of the protein is greater than approximately 98%. The yields of target protein can be up to 30 mg/L. Crystals with better shape were obtained with reservoir solution consisting of 0.2 M MgSO4.6 H2O, 20% w/v PEG3350.