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首页> 外文期刊>Protein Expression and Purification >Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)
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Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)

机译：甲基营养酵母巴斯德毕赤酵母（Pichia pastoris）作为宿主，用于从马铃薯块茎（Solanum tuberosum）生产ATP-二磷酸水解酶（apyrase）

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ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1 mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48 kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 41]
机译：在二价阳离子存在的情况下，ATP-二磷酸水解酶（apyrase）催化三磷酸和二磷酸核苷的磷酸酐键水解。该酶对核苷酸具有广泛的底物特异性，这使其成为不同生物技术应用（例如DNA测序和血小板聚集抑制）的理想酶。从马铃薯块茎中分离出唯一可商购的腺苷三磷酸双磷酸酶。为了避免批次和批次之间的活性和质量差异，我们决定生产一种重组酶。选择了甲基营养酵母巴斯德毕赤酵母作为真核表达宿主。将没有信号肽的马铃薯腺苷三磷酸酶的编码序列克隆到YpDC541载体中，以与酿酒酵母的α-交配分泌信号融合。将该基因置于甲醇诱导的醇氧化酶启动子的控制下。 YpDC541-apyrase构建体被整合到巴斯德毕赤酵母菌株SMD1168中。甲醇诱导导致腺苷三磷酸腺苷酶的分泌达到1 mg / L。通过疏水相互作用和离子交换色谱法纯化具有生物活性的重组腺苷三磷酸双磷酸酶。根据SDS-PAGE和蛋白质印迹分析，纯化的酶显示为高糖基化的。通过酶促除去N-聚糖，检测到对应于48kDa分子量的单个条带。当与焦磷酸测序技术结合使用时，发现重组腺苷三磷酸腺苷酶功能良好。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：41]

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