Vasu Prasanna, Bauer Stefan, Savary Brett J
Food Safety and Analytical Quality Department, Central Food Technological Research Institute (CSIR), Cheluvamba Mansion, Mysore, India.
Methods Mol Biol. 2012;824:393-416. doi: 10.1007/978-1-61779-433-9_21.
The methylotrophic yeast Pichia pastoris is increasingly used for heterologous expression of high quality proteins in laboratory-scale (milligram) quantities. Commercially available polysaccharide-active enzyme preparations have limited applications in plant cell wall research due to their heterogeneous mix of hydrolytic activities. P. pastoris provides an ideal in vitro expression system for producing monocomponent enzymes, since it lacks endogenous plant cell wall-active enzymes and can perform eukaryotic post-translational modifications (i.e., glycosylation). We have routinely prepared cDNA constructs from Aspergillus nidulans encoding a broad array of hydrolases active on various linkages contained in plant cell wall polysaccharides. The cDNAs were inserted into the pPICZα C shuttle vector (Invitrogen) in-frame with the Saccharomyces cerevisiae α-secretion factor and expressed under the transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) promoter. The enzyme products were efficiently secreted into buffered complex methanol medium (BMMY) as C-terminal his-tagged proteins for simple one-step affinity purification. The insertion of the c-Myc epitope enabled easy immunodetection. Here we present the detailed protocols for primer design, cloning, expression, and activity assays for a representative set of xylan-acting hemicellulases produced in P. pastoris.
甲基营养型酵母巴斯德毕赤酵母越来越多地用于在实验室规模（毫克级）大量异源表达高质量蛋白质。市售的多糖活性酶制剂由于其水解活性的异质混合，在植物细胞壁研究中的应用有限。毕赤酵母提供了一个理想的体外表达系统来生产单组分酶，因为它缺乏内源性植物细胞壁活性酶，并且可以进行真核生物的翻译后修饰（即糖基化）。我们常规地从构巢曲霉制备了编码对植物细胞壁多糖中各种键具有活性的多种水解酶的cDNA构建体。将这些cDNA与酿酒酵母α分泌因子读框内插入pPICZα C穿梭载体（Invitrogen），并在高度诱导型醇氧化酶1（AOX1）启动子的转录控制下表达。酶产物作为C端带有组氨酸标签的蛋白质有效地分泌到缓冲复合甲醇培养基（BMMY）中，用于简单的一步亲和纯化。插入c-Myc表位便于进行免疫检测。在此，我们展示了在毕赤酵母中产生的一组代表性木聚糖作用半纤维素酶的引物设计、克隆、表达和活性测定的详细方案。
