High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris
High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris
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DOI：
10.1016/j.pep.2004.09.009
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发表时间：
2005-01-01
影响因子：
1.6
通讯作者：
Kitabatake, N
中科院分区：
文献类型：
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作者：
Masuda, T;Ueno, Y;Kitabatake, N
鸡蛋溶菌酶(HEL)是一种甜味蛋白质。来理解为什么溶菌酶是甜的。用重组方法高产率地合成了该酶。将成熟的HEL基因从Taq聚合酶扩增产物克隆到巴斯德毕赤酵母表达分泌载体pPIC6pha中。该表达载体同时含有酿酒酵母α-前交配因子分泌信号和BSD基因，用于细菌和酵母转化子的筛选。在发酵罐中进行HEL的表达。培养上清液经超滤浓缩、CM-离子交换层析纯化。获得了约400mgL(-1)的重组HEL。重组溶菌酶的高产量使我们能够在人类身上进行感官分析。纯化的重组溶菌酶和直接从蛋清中纯化的溶菌酶一样具有甜味，并显示出对黄色微球菌细胞的完全裂解活性。这些结果表明，带有杀菌素S选择系统的巴斯德毕赤酵母表达系统可以高产率地生产活性形式的重组甜味蛋白。(C)2004 Elsevier Inc.保留所有权利。
Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet. the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia Pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL(-1) of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield. (C) 2004 Elsevier Inc. All rights reserved.