黑曲霉糖化酶基因在毕赤酵母X33中的高效表达

为高效表达黑曲霉糖化酶基因,根据GenBank中糖化酶的氨基酸序列(登录号:AY652617),选用毕赤酵母(Pichia pastoris)密码子偏嗜性,全基因合成新的cDNA序列。改造后的基因克隆到pGAPZαA质粒中,获得重组分泌型酵母表达质粒pGAPZαA-EC,经限制性内切酶Bln Ⅰ酶切线性化后,电击转化入毕赤酵母细胞X33内。经高浓度博莱霉素抗性筛选,得到高拷贝转化子,PCR检测结果显示,糖化酶基因与毕赤酵母染色体稳定结合。糖化酶蛋白获得分泌表达,SDS-PAGE分析其分子质量约为80 ku,其表达量约为180 mg/L。表达产物经Starch-PAGE活性染色,显示其具有酶学活性。

High Expression of Glucoamylase Gene of Aspergillus niger in Pichia pastoris X33

In order to improve expression of glucoamylase in Pichia pastori,based on the gene sequences encoding as registered in GenBank(serial number is AY652617),using the partiality condon of P.pastoris,the gene was designed and synthesized.The modified gene was cloned into the pGAPZαA vector to construct the recombinant expression vector pGAPZαA-EC.Then the pGAPZαA-EC which was linearized by Bln Ⅰ was transformed into P.pastoris X33 by electroporation.The transformants were screened with Zeocin and multiply-copy colonies were harvested,in which glucoamylase gene was verified to be inserted into yeast chromosome stably.SDS-PAGE result showed that,a 80 ku secreted protein was produced,consistent with the expected one,concentration of which was 180 mg/L in supernatant.Expression product showed enzymatic activity by Starch-PAGE.