草酸青霉16β-葡萄糖苷酶的异源表达

The Heterologous Expression of Penicillium oxalicum 16β-Glucosidase

为探究真核菌株草酸青霉16的β-葡萄糖苷酶(16BGL)在不同宿主中的异源表达情况,该文通过基因克隆技术,将16BGL基因分别与pPIC9K、pET-28a、pGAPZαA载体进行连接,转化到毕氏酵母GS115或大肠杆菌Rosetta(DE3)上,筛选阳性克隆子,比较并分析其表达情况.结果表明:16BGL-9K未能成功电转到毕氏酵母GS115上;将16BGL-28a转化到大肠杆菌Rosetta上,使用最终物质的量浓度为50μmol·L^(-1)的IPTG和质量分数为1.7%的乳糖在22℃下诱导可实现高水平表达,但极易形成包涵体且无活性.为了消除包涵体的影响,在相同条件下将16BGL的N端加入DsbA分泌信号肽,经SDS-PAGE检验,16BGL有高水平可溶性表达,但无活性;将16BGL-ZαA电转到GS115上,筛选阳性克隆子并接种到含YPG培养基的摇瓶中,结果表明:上清液蛋白质量浓度达到23.723 mg·mL^(-1),且有活性.真核来源的16BGL基因适合于真核宿主表达.

The heterologous expression of β-glucosidase(16BGL)of a eukaryotic strain Penicillium oxalicum 16 in different hosts is explored.The 16BGL gene is ligated with pPIC9K,pET-28a,and pGAPZαA respectively,and transformed into Pichia pastoris GS115 or Escherichia coli Rosetta(DE3),the positive clones are screened,and their expression is compared and analyzed.16BGL-9K can not be successfully transferred into GS115 by electroporation.16BGL-28a in Rosetta achieves a high level of expression after induction at 22℃ with IPTG at a final concentration of 50μmol·L^(-1) and 1.7% lactose,but it is very easy to form inclusion bodies without activity.In order to eliminate the influence of inclusion bodies,a secretion signal peptide DsbA is added in the N-terminal of 16BGL,16BGL achieves a high level of soluble expression under the same conditions,but no activity is detected by SDS-PAGE.16BGL-ZαA is transferred to GS115,the positive clones are screened and inoculated into YPG,and the supernatant possesses a protein concentration of 23.723 mg·mL^(-1) and shows an enzymatic activity.The eukaryotic gene 16BGL is suitable for expression in eukaryotic hosts.