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首页> 外文期刊>Protein Expression and Purification >Expression, purification, and characterization of an unstable lysozyme mutant in Pichia pastoris
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Expression, purification, and characterization of an unstable lysozyme mutant in Pichia pastoris

机译：巴斯德毕赤酵母中一个不稳定的溶菌酶突变体的表达，纯化和鉴定

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摘要

To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 20]
机译：为了研究不稳定的异源蛋白在巴斯德毕赤酵母中的表达和纯化，整合了H5-溶菌酶的cDNA，H5溶菌酶是一种带有疏水性五肽（Phe-Phe-Val-Ala-Pro）融合在羧基末端的鸡蛋溶菌酶突变体。进入巴斯德毕赤酵母的基因组。发现培养基组成，诱导时间和发酵罐类型是H5-溶菌酶表达的重要因素。当将α-因子的前原序列用作分泌信号序列时，通过用甲醇诱导来分泌基本上活性的H5-溶菌酶。分泌的量比酿酒酵母所观察到的高422倍。直接从发酵液中回收重组H5-溶菌酶并通过阳离子交换色谱法纯化。突变的溶菌酶对革兰氏阳性菌和革兰氏阴性菌均表现出杀菌活性。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：20]

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