铜绿假单胞菌丙酸代谢关键酶基因的克隆表达及生物信息学分析

Cloning,expression and bioinformatic analysis of propionate metabolism key enzyme genes from Pseudomonas aeruginosa

目的克隆表达丙酸代谢关键酶基因prpB、prpC和prpD,分析预测相应表达蛋白潜在的结构和功能,为铜绿假单胞菌新型抗菌靶点的开发奠定基础。方法对铜绿假单胞菌中丙酸代谢关键基因prpB、prpC、prpD克隆入大肠埃希菌,构建相应的基因工程重组菌,通过镍柱亲和层析纯化重组蛋白,SDS-PAGE检测PrpB、PrpC和PrpD蛋白的分子质量及纯度。利用生物信息学软件预测PrpB、PrpC和PrpD蛋白的理化性质、疏水性、信号肽、二级结构和三级结构。结果重组菌用0.5 mmol/L IPTG于16℃条件下诱导,表达的重组蛋白PrpB、PrpC和PrpD主要以可溶性形式存在于上清中,经Ni柱亲和层析纯化得到相对分子质量分别为32×10^(3)、42×10^(3)、55×10^(3)的目的蛋白,与预期一致。生物信息学分析PrpB、PrpC和PrpD蛋白分别由298、375、494个氨基酸组成,相对分子质量分别为32.14×10^(3)、41.69×10^(3)、54.88×10^(3),等电点分别为5.33、6.03、6.15。3种蛋白均为亲水性蛋白,无信号肽,无跨膜结构。蛋白亚细胞定位于细胞质中,α螺旋和无规则卷曲是PrpB、PrpC和PrpD二级结构的主要蛋白质元件。结论成功表达了重组蛋白PrpB、PrpC和PrpD并进行了纯化。生物信息学预测PrpB、PrpC和PrpD含有丰富的α螺旋和无规则卷曲结构,为揭示PrpB、PrpC和PrpD蛋白的结构及丙酸代谢在铜绿假单胞菌中的生物学功能奠定了理论基础。

Objective To clone and express prpB,prpC and prpD genes of key enzymes of propionate metabolism,further analyze and predict PrpB,PrpC and PrpD potential structures and functions and lay the foundation for developing new antimicrobial targets of Pseudomonas aeruginosa.Methods The prpB,prpC and prpD of key genes in propionate metabolism from P.aeruginosa were cloned into Escherichia coli BL21(DE3).The recombinant strains of corresponding genes were constructed and the recombinase were purified with Ni-NTA column affinity chromatography.The molecular weight and purity were determined by SDS-PAGE.Meanwhile,bioinformatics software is employed for detecting the physicochemical properties,hydrophobicity,signal peptide,secondary structure and tertiary structure of PrpB,PrpC and PrpD proteins.Results The recombinant bacteria were induced with 0.5 mmol/L at 16℃overnight.The recombinant proteins PrpB,PrpC and PrpD mainly existed in the form of soluble supernatant.The PrpB,PrpC and PrpD protein,which relative molecular weight was 32×10^(3),42×10^(3) and 55×10^(3) respectively,were purified by Ni-NTA affinity chromatography.PrpB,PrpC and PrpD protein with molecular weights of 32.14×10^(3),41.69×10^(3),54.88×10^(3) were composed of 298,375 and 494 amino acids respectively,and isoelectric point is 5.33,6.03 and 6.15;PrpB,PrpC and PrpD protein lacked signal peptide and transmembrane structure is featured by the hydrophilic protein;PrpB,PrpC and PrpD protein localized in the cytoplasm.Andαhelix and random coil play the main role in constructing protein elements of the secondary structure of PrpB,PrpC and PrpD protein.Conclusion The recombinant protein PrpB,PrpC and PrpD were successfully induced expressed and purified.The results of bioinformatics demonstrated that PrpB,PrpC and PrpD protein are rich inαhelix and irregular curly structures.The study lays a theoretical foundation for revealing the structures of PrpB,PrpC and PrpD protein and the biological functions of propionate metabolism in P.aeruginosa.