大蒜蒜氨酸酶基因的克隆及在大肠杆菌中的表达

Cloning of Alliinase Gene from Garlic And Its Expression in Escherichia coli

提取大蒜总RNA,经RT-PCR扩增出1348bp的成熟蒜氨酸酶基因,克隆到pET-28a(+)载体中,构建带有寡聚组氨酸纯化标签的融合蛋白表达质粒。将重组质粒转化入大肠杆菌,经IPTG诱导后的表达产物大部分为包含体,SDS-PAGE电泳和western blotting结果表明目标蛋白的相对分子质量约5.3×104,占菌体总蛋白的45.1%。含6mol/L盐酸胍的溶液,经镍亲和色谱纯化和透析复性,每升摇瓶发酵液可得蛋白143.3mg,得率为13.6%,纯度为97%,蒜氨酸酶的比活力为388u/mg。

Total RNA from garlic bulbs was extracted.The 1 348 bp gene of mature alliinase was amplified by RT-PCR and inserted into pET-28a（＋） to construct an alliinase expression plasmid followed by transformation to E.coli BL21（DE3）.The recombinant protein,mainly in form of inclusion body,with a molecular weight of about 5.3×104,was successfully expressed by IPTG induction and accounted for 45.1% of total protein.After the inclusion body was dissolved by the solution containing 6 mol/L guanidine hydrochloride and protein was purified by immobilized Ni2＋ affinity chromatography,13.6% of the recombinant protein was recovered with 97% purity.143.3 mg Protein was obtained from one liter of fermentation broth in shake flask.Purified protein could catalyze alliin to pyruvate and its relative activity was 388 u/mg.