变形假单胞菌2-酮基葡萄糖酸差向异构酶基因的原核表达

采用降落聚合酶链式反应(touchdown polymerase chain reaction,TD-PCR)技术,从2-酮基葡萄糖酸工业生产菌株变形假单胞菌JUIM01中克隆了编码2-酮基葡萄糖酸差向异构酶的基因kgu E。将目的基因与质粒pET-28a(+)重组后转入宿主表达菌中,获得了重组菌株大肠杆菌BL21(DE3)/pET-28a(+)-kgu E。在异丙基-β-D-硫代半乳糖苷的诱导下,该重组菌株表达了一个分子质量约为30.5 ku的融合蛋白,且该蛋白的Western-blot鉴定结果显示为阳性。生物信息学分析结果表明,该蛋白是一种由256个氨基酸残基组成的分子质量为28.5 ku的亲水性蛋白,与恶臭假单胞菌的2-酮基葡萄糖酸差向异构酶在氨基酸序列上的一致性达78%,定位于细胞质中,其二级结构中α-螺旋、延伸链和无规则卷曲所占的比例分别为40.62%、17.19%和42.19%。本研究结果为变形假单胞菌2-酮基葡萄糖酸差向异构酶的功能研究提供了一定理论支持。

Prokaryotic Expression of a 2-Ketogluconate Epimerase Gene from Pseudomonas plecoglossicida

The complete nucleotide sequence of the gene (kguE) encoding 2-ketogluconate epimerase was cloned by touchdown polymerase chain reaction (TD-PCR) from an industrial 2-ketogluconic acid producer, Pseudomonas plecoglossicida JUIM01. A recombinant vector was constructed by ligating the restriction enzymes digested products of the target gene to pET-28a(+) vector and then transferred into the expression host E. coli BL21(DE3). A specific fusion protein with molecular weight of about 30.5 ku was expressed in the recombinant strain E. coli BL21(DE3)/pET-28a(+)-kguE after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction with a positive Western-blot result. Bioinformatic analysis showed that the protein was predicted to be a hydrophilic protein with molecular weight of 28.5 ku located in the cytoplasm, sharing 78% amino acid sequence identity with the 2-ketogluconate epimerase from P. putida. The predicted secondary structure consisted of 40.62% of α-helix, 17.19% of extended strand and 42.19% of random coil. This study is expected to provide a basis for further elucidating the function of 2-ketogluconate epimerase in P. plecoglossicida.