摘要
化脓性链球菌是一种革兰氏阳性人类致病菌,其生长和感染离不开铁离子。MtsA是化脓链球菌中直接结合铁离子的一个脂蛋白。通过PCR扩增化脓性链球菌MGAS5005中的MtsA基因,构建高效表达质粒pGEX-MtsA,将其转化到大肠杆菌BL21中并用IPTG进行诱导表达。利用亲和层析方法纯化表达产物。通过多序列比对分析了MtsA铁结合中心的保守性,利用定点突变技术将MtsA的结合配体单位点及多位点进行突变,结合圆二色谱分析这些氨基酸突变后蛋白二级结构的变化。多序列比对结果表明MtsA结合铁离子的氨基酸位点是保守的,4个关键氨基酸位点处于蛋白空间的凹陷处。通过比较空载及饱和铁离子的野生型蛋白以及突变体蛋白的圆二色吸收光谱,发现野生型MtsA结合铁离子后结构更加紧密,H68A,E206A和D281A则比野生型的二级结构松散,而H140A的二级结构和野生型的几乎没有差别。对MtsA 4个结合配体突变后其二级结构变化的研究,为进一步研究细菌中的铁转运机理及开发疫苗候选药物和药靶奠定了一定的理论依据。
Streptococcus pyogenes is a Gram-positive human pathogen, and iron is essential for its survival and infection. MtsA is a lipoprotein of Streptococcus pyogenes, which is responsible for iron binding. MtsA was amplified by PCR from Streptococcus pyogenes MGAS5005 and constructed the recombinant plasmid pGEX-MtsA. The recombinant plasmid was transformed into Escherichia coli BL21 to express the fusion protein after induction with IPTG. The protein was purified using affinity chromatography. The conservative of the MtsA iron binding center was analyzed using multiple alignment. The mutant proteins were constructed by site-directed mutagenesis. Circular dichroism was used to collect the changes of mutants＇ secondery structure when compared to wild-type protein. The result of multiple alignment showed the four binding amino acids were conserved and were in the hollow of MtsA space structure. The CD spectra of WT MtsA and mutants were collected respectively. The a-helix content increased when Fe2＋ was added to the apo-protein solution, which indicated that the metal binding induced some conformational change. For the apo mutant proteins H68A, E206A and D281A, the contents of a-rhelix more or less decreased, reflecting that mutations caused alterations of the secondary structures at some extends but not substantially disturbed the protein conformation. The secondary-structural change in mutant H140A was unexpectedly barely detectable when compared to WT MtsA. These results provided a valuable information for the understanding of iron transport in bacteria, which may be helpful for the development of novel strategies in the control of bacterial infection.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2012年第12期13-19,共7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
关键词
化脓链球菌MtsA定点突变
多序列比对
圆二色谱法
Streptococcus pyogenesdichroismMtsA Site-directed mutagenesis Muhiple alignment Circular
