荧光素酶表达载体构建及其在巴斯德毕赤酵母的表达

萤火虫荧光素酶(Firefly Luciferase,FL)是ATP快速检测技术的核心组件,通过荧光强度与ATP浓度的对应关系,在食品行业检测微生物数量发挥着重大作用。本文为实现荧光素酶在重组毕赤酵母GS115中的异源表达,通过从载体pGL2-control中扩增荧光素酶基因luc,克隆到真核表达载体p PIC9K中,线性化后电击转化毕赤酵母GS115菌株,筛选阳性重组菌株。在甲醇的诱导下进行酶的表达,对粗酶液进行生物活性发光分析,然后对粗酶进行超滤、阴离子层析和分子筛凝胶层析三步纯化。甲醇诱导表达96 h发现胞外和胞内粗酶液均有相对较高的酶发光活性,酶活分别为1.45×10~6 RLU/mL和1.58×10~9 RLU/mL。SDS-PAGE与Western blot分析重组荧光蛋白大小约为70 ku,最终纯化得到的荧光素酶,其比活为7.0×10~8 RLU/mg,纯化倍数达到19.3倍,产量为48 mg/L。以上结果表明,荧光素酶能够在毕赤酵母表达系统获得较好的表达和纯化效果。

Construction of Luciferase Expression Vector and Its Expression in Pichia pastoris

Firefly Luciferase (Fireflyluciferase, FL) is the core component of ATP bioluminescence assay, which plays a critical role in the detection of foodborne microorganisms in food industrythrough the correlation between the luminous intensity and ATP concentrations ., In order to realize the heterologous expression of luciferase in recombinant P. pastoris GS115, the luciferase gene was amplified and cloned into the eukaryotic expression vector pPIC9K. And then the vector was linearized and was transformed into the strain GS115 by electricity to screen the positive transformants. Later ATP bioluminescence assay was employed to test the enzyme activity of both extracellular and intracellular crude enzyme liquid following the expression induced by methanol. The crude enzyme was then purified by ultrafiltration, anion exchange and size exclusion chromatography sequentially. The extracellular and intracellular crude enzyme liquid all had relatively higher enzyme activity after a 96 h-methanol induction, which were 1.45×106 RLU/mL and 1.58×109 RLU/mL, respectively. SDS-PAGE and westernblot analysis indicated that the protein size of FL was about 70 ku. The purified luciferase activity was 7.0×108 RLU/mg with the purification fold of 19.3 and the yield of 48 mg/L. In conclusion, our study here demonstrated that the firefly luciferase could be well expressed and purified in the eukaryotic expression system as P. pastoris GS115.