肺炎链球菌HMG-CoA合成酶基因克隆和表达

Cloning and expression of the mvaS from Streptococcus pneumoniae

目的克隆、表达肺炎链球菌3-羟基-3-甲基戊二酰辅酶A合成酶(3-hydroxy-3-methylglutaryl-coenzyme Asynthase,HMGS)。方法采用基因工程技术克隆肺炎链球菌HMGS基因,对T226进行反突变,构建重组表达载体pET-HMGSm并表达HMGS。结果序列分析表明该基因与文献中报道的肺炎链球菌HMGS基因(No.AF290098)相比较有98.5%的同源性,存在18个核苷酸的差异,其中有2个核苷酸的不同导致编码的氨基酸改变,分别是Asn259→Ser,Ala349→Val,该基因编码蛋白由398个氨基酸组成。结论优化重组HMGS表达条件,在18℃0.4 mmol/L IPTG诱导表达4 h,用Ni-NTA层析柱分离纯化得到46 kDa特异性蛋白。

Objective To clone and express 3-hydroxy-3-methylglutaryl-coenzyme A synthase（HMGS） from Streptococcus pneumoniae.Methods The mvaS was cloned from S.pneumoniae.The nucleotide mutated（A226→T） was amplified to conduct back mutation.The recombinant expression plasmid pET-HMGSm was constructed and expressed.Results In comparison with the mvaS sequence of S.pneumoniae published in NCBI（AF290098）,there were 98.5% homology and 18 nucleotides had been changed,resulting in two changed amino acids（Asn259→Ser,Ala349→Val）.The HMGS of S.pneumoniae consisted of 398 amino acid residues.Conclusion The recombinant plasmid pET-HMGSm was expressed with 0.4 mmol/L IPTG induction for 4 h at 18 ℃,the product analyzed by SDS-PAGE showed that a specific protein band appeared with a molecular weight about 46 kDa.