嗜酸乳杆菌ATCC4356分选酶A基因克隆、表达及功能分析

分选酶在细菌对宿主细胞的黏附过程中具有重要作用。为探究乳酸菌分选酶的特性及与其他致病菌分选酶的差异,以嗜酸乳杆菌ATCC4356为模板,构建分选酶A(sortase A,SrtA)重组表达载体p ET28a-SrtA_(ΔN48),转入大肠杆菌感受态细胞Transetta(DE3)内表达,使用Dabcyl-QALPTTGEE(Edans)探究其酶活特性。结果发现:嗜酸乳杆菌ATCC4356 SrtA(Lap-SrtA_(ΔN48))分子质量为20 k Da左右;Mg~(2+)、Zn~(2+)、Mn~(2+)可提高Lap-SrtAΔN48的转肽能力;Ca~(2+)不会影响SrtAΔN48的活性,明显区别于致病菌;且分选酶抑制剂查尔酮会抑制Lap-SrtA_(ΔN48)的活性。进一步通过蛋白质同源建模分析发现Lap-SrtA由8条中心β桶状结构互相折叠而成,属于经典的分选酶结构,Lap-SrtA的活性位点为His137、Cys198、Arg205。

Cloning,Expression and Characterization of Sortase A from Lactobacillus acidophilus ATCC4356

Sortase (Srt) plays an important role in the adhesion of Gram-positive bacteria to host cells. The focus of this study was to recombine the sortase A (SrtA) of Lactobacillus acidophilus (Lap-SrtA) in E. coli and to evaluate the differences between Lap-SrtA with other pathogenic sortase. The srtA gene was cloned from L. acidophilus ATCC4356 and expressed in E. coli Transetta (DE3). The recombinant protein was purified and analyzed using Dabcyl-QALPTTGEE (Edans) as substrate. The results showed that the molecular weight of the recombinant protein was about 20 kDa with an Lap-SrtA activity, and the activity could be enhanced by Mg2+, Zn2+, and Mn2+. However, Ca2+ had no visible effect on the activity of SrtA, making it distinct from other pathogenic sortase. Chalcone could significantly inhibit SrtA activity. Furthermore, there were eight segments of β sheet structure in Lap-SrtA as analyzed by homology modeling using SWISS MODEL, and the active site residues of Lap-SrtA were found to be His 137, Cys 198 and Arg 205.