克雷伯肺炎杆菌1,3-丙二醇氧化还原酶基因克隆及表达条件研究

Cloning of 1,3-propanediol oxidoreductase gene from Klebsiella pneumoniae and preliminary study on its expression conditions

1,3-丙二醇氧化还原酶是甘油歧化为1,3-丙二醇的一种关键酶。本研究从克雷伯肺炎杆菌(Klebsiella pneumoniae)基因组中,用PCR方法克隆了其编码基因dhaT。TA克隆测序正确后,构建胞内表达载体pET-28a-dhaT和分泌表达载体pET-22b-dhaT,然后转化E.coliBL21(DE3)进行原核表达。表达部位确定、SDS-PAGE和酶活分析表明,该酶得到了高水平表达。其中使用pET-22b表达的目的蛋白大都是不溶的包涵体;而使用pET-28a表达的目的蛋白胞内可溶,占胞内可溶总蛋白的45%,占菌体总蛋白的25%。常规(30℃)诱导表达即呈现1,3-丙二醇氧化还原酶活性,但低温(20℃)14 h诱导显示3.7倍的酶活性。

1,3-propanediol oxidoreductase （PDOR） is one of the two key enzymes responsible for conversion of glycerol to 1,3-propanediol. In this study, its encoding gene dhaT was cloned by PCR from the genome of K, pneumoniae and inserted into pMD18-T. After DNA sequencing, both prokaryotic expression vectors, pET-28a-dhaT and pET-22b- dhaT were constructed respectively. Subsequently, recombinant plasmids were transformed into E. coli strain BL21 （DE3） and the transformed strain was induced with IPTG. The results of SDS-Page analysis and protein localization assay reflected the high expression of PDOR in E. coli EL21 （DE3）. Of the two recombinant strains, E. coli EL21 （DE3） （pET-28a-dhaT） could highly express soluble PDOR, accounting for 45 % of total soluble protein in cytoplasm or 25 % of total protein in the host. In contrast, almost all PDOR expressed by E. coli 13L21（DE3） （pET-22b-dhaT） was insoluble inclusion body. Besides, the effect of induction temperature on enzyme activity was also investigated. Although PDOR expressed at normal temperature （30 ℃ ） showed certain enzyme activity, it displayed 3.7 times activity when induced at lower temperature （20 ℃ ）, suggesting the distinct effect of temperature on protein expression. Furthermore, the advantages and disadvantages of both expression vectors were also discussed in this paper.