海藻糖合成酶基因的克隆及原核表达

Cloning and prokaryotic expression of trehalose synthase gene

目的克隆海藻糖合成酶(Trehalose synthase,Tres)基因,并在大肠杆菌中表达重组酶。方法以长白山温泉Thermus thermophilus SH-110基因组DNA为模板,PCR扩增Tres基因,克隆至原核表达载体pET-30a(+)中,构建重组表达质粒pET-30a(+)-Tres,转化E.coli BL21(DE3),分别采用IPTG和乳糖诱导表达,并对表达产物进行SDS-PAGE、Western blot和薄层色谱(Thin layer chromatography,TLC)分析。结果重组表达质粒pET-30a(+)-Tres经双酶切及测序证实构建正确;IPTG和乳糖均能诱导重组蛋白的表达,且乳糖诱导的蛋白表达量较高;表达的重组酶Tres相对分子质量约为110 000,可与鼠抗6×His单克隆抗体特异性结合;粗酶液的酶活力为8 760 U/μg,是野生菌酶活的10倍;重组酶水解麦芽糖产物经TLC分析证实,其具有较强的海藻糖合成酶活性。结论已成功在大肠杆菌中表达了重组Tres,为大规模生产海藻糖奠定了基础。

Objective To clone trehalose synthase （Tres） gene and express in E. coli. Methods Tres gene was amplified by PCR using the genomic DNA of Thermus thermophilus SH-110 as a template, and cloned into prokaryotic expression vector pET- 30a （＋）. The constructed recombinant plasmid pET-30a （＋）-Tres was transformed to E. coli BL21 （DE3） and induced with IPTG and lactose respectively. The expressed product was identified by SDS-PAGE, Western blot and thin layer chromatography （TLC）. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-30a（＋）-Tres was constructed correctly. Both IPTG and lactose induced high expression of recombinant protein. The activity of crude Tres, with a relative molecular mass of about 110 000, was 10 times of that of wild The rmus the rmophilus SH-110. The recombinant protein showed high activity of Tres as proved by TLC analysis of hydrolysate of maltose. Conclusion Recombinant Tres was successfully expressed in E. coli, which laid a foun- dation of large-scale production of trehalose.