aiiA基因表达载体的构建及其在毕赤酵母中的表达

Construction of an aiiA-expressing Vector and Its Expression in Pichia pastoris

摘要:

构建携带N-酰基高丝氨酸内酯酶基因(aiiA)的重组毕赤酵母表达载体pPIC3.5K-aiiA, 采用电转化方法转入毕赤酵母GS115, 经营养缺陷型培养基、表型鉴定和高G418浓度筛选获得高拷贝表达盒的酵母转化子, 用0.5%甲醇诱导表达, RT-PCR鉴定可检测到重组酵母中编码目的基因成熟肽的mRNA, SDS-PAGE和Western blot检测结果表明, aiiA基因在毕赤酵母中成功表达, 用指示菌紫色杆菌CV026检测发现目的蛋白具有降解N-酰基高丝氨酸内酯的活性。

Abstract:

An aiiA-expressing vector, pPIC3.5K-aiiA, was constructed and transformed into Pichia pastoris GS115 by electroporation. The recombinant yeast strains were screened with auxotroph medium and phenotypic identification.The transformants with high copy numbers of aiiA gene were selected in medium containing high concentration of G418. The expression of aiiA was induced by addition of methanol into culture at the final concentration of 0.5%. The transcription of aiiA was confirmed by RT-PCR in the recombinant yeast. SDS-PAGE and Western blot analysis demonstrated that recombinant AiiA protein was successfully expressed after induction. The recombinant AiiA protein showed the activity of degrading the N-acyl-homoserine lactones when using Chromobacterium violaceum CV026 as reporter strain.