肺炎链球菌去信号肽脂蛋白PsaA锌离子结合特性的初步表征  

Preliminary Characterization of a Streptococcus pneumoniae Lipoprotein PsaA Without Signal Peptides for Zinc-binding Properties

作　　者：李楠[1] 王南杰[1] 王红翠[1] 李慧[1] 詹胜[1] 阳小燕[1] 孙雪松[1] 

机构地区：[1]暨南大学生命与健康工程研究院,广州510632

出　　处：《中国生物工程杂志》2011年第12期27-32,共6页China Biotechnology

基　　金：国家自然科学基金(31000373);广东省自然科学基金(10451063201005247)资助项目

摘　　要：肺炎链球菌是细菌性肺炎的主要病原体。PsaA是各种肺炎链球菌共有的遗传保守的特异性表面金属结合脂蛋白。通过PCR扩增肺炎链球菌D39不含信号肽的PsaA基因片段,将其通过T4连接酶连接至含6His标签的表达载体PBAD/HisA中,转化表达宿主大肠杆菌Top-10后用L(+)-阿拉伯糖诱导重组蛋白的表达。重组蛋白经亲和镍柱纯化以后,用外切酶-重组肠激酶(REK)去除6His标签。感应偶合电浆质谱(ICP-MS)测得纯化的PsaA蛋白以1∶1比例结合金属锌离子。进而,通过圆二色谱法分析金属离子的结合对蛋白二级结构中α-螺旋和β-片层含量的影响,荧光光谱研究蛋白结合锌离子的解离常数及结合当量,为进一步研究该蛋白在体外的金属结合特性及细菌的金属运输及毒力机制提供理论基础。Streptococcus pneumoniae is a major pathogen of bacterial pneumonia.PsaA is a surface metal-binding lipoprotein and genetically conserved in all kinds of Streptococcus pneumoniae.psaA gene was amplified by polymerase chain reaction（PCR） from Streptococcus pneumoniae D39 and cloned into the expression vector PBAD / HisA containing 6His tag.The recombinant plasmid was transformed into Escherichia coli Top-10 to express the fusion protein after induction with L（＋） arabinose.The recombinant protein was further purified by a nickel affinity column and the 6His tags were removed with recombinant enterokinase.Inductively coupled plasmamass spectrometry（ICP-MS） analysis revealed that the purified PsaA binds Zn2＋ in a ratio of 1∶1.Circular dichroism showed that zinc binding did not induce changes of the α-helix and β-sheet content in PsaA.Fluorescence spectra determined the Zn2＋-binding affinity and the dissociation constant（Kd） which was calculated to be 1.19058 μmol.These results are useful for the further study on the metal-binding properties of PsaA protein in vitro,bacterial metal transport and virulence mechanism of Streptococcus pneumoni.

关 键 词：肺炎链球菌 PSAA 圆二色谱法 ICP-MS 荧光光谱 

分 类 号：Q819[生物学—生物工程]