Weidner Maria, Taupp Marcus, Hallam Steven J
Department of Microbiology and Immunology, University of British Columbia.
J Vis Exp. 2010 Feb 25(36):1862. doi: 10.3791/1862.
Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZalphaA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the alpha-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.
在真核微生物宿主毕赤酵母中进行蛋白质表达，为在快速且易于使用的表达系统中大量生产重组蛋白提供了可能。作为单细胞微生物，毕赤酵母易于操作，能在廉价培养基上以高细胞密度快速生长。作为真核生物，毕赤酵母能够进行高等真核细胞所进行的许多翻译后修饰，所获得的重组蛋白会经历蛋白质折叠、蛋白水解加工、二硫键形成和糖基化[1]。作为甲基营养型酵母，毕赤酵母能够将甲醇作为唯一碳源进行代谢。醇氧化酶AOX1的强启动子受到严格调控，并由甲醇诱导，它被用于目的基因的表达。因此，通过向生长培养基中添加甲醇可诱导外源蛋白的表达[2; 3]。另一个重要优势是利用信号序列将重组蛋白分泌到生长培养基中，该信号序列可将外源蛋白靶向至毕赤酵母的分泌途径。由于酵母自身分泌到培养基中的内源蛋白水平较低，且培养基中未添加其他蛋白，因此异源蛋白构成了培养基中总蛋白的大部分，便于后续的蛋白纯化步骤[3; 4]。此处使用的载体（pPICZalphaA）包含用于严格调控、甲醇诱导目的基因表达的AOX1启动子；用于分泌重组蛋白的α因子分泌信号；用于在大肠杆菌和毕赤酵母中进行筛选的博来霉素抗性基因；以及一个包含c-myc表位和多组氨酸（6xHis）标签的C末端肽，用于检测和纯化重组蛋白。我们还使用识别亲本载体上c-myc表位的特异性抗myc-HRP抗体，对重组蛋白进行了蛋白质印迹分析。
