Masuda Tetsuya, Ueno Yuki, Kitabatake Naofumi
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Protein Expr Purif. 2005 Jan;39(1):35-42. doi: 10.1016/j.pep.2004.09.009.
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.
鸡蛋清溶菌酶（HEL）是一种具有甜味的蛋白质。为了弄清楚溶菌酶为何具有甜味，采用重组方法以高产率合成了该酶。将成熟的HEL基因从Taq聚合酶扩增的PCR产物克隆到巴斯德毕赤酵母表达和分泌载体pPIC6α中。该表达载体同时包含酿酒酵母前原α-交配因子分泌信号和杀稻瘟菌素抗性基因（bsd），用于在细菌和酵母中筛选转化体。在发酵罐培养物中进行HEL的表达。通过超滤浓缩培养上清液，并通过CM离子交换色谱法进行纯化。获得了约400mg/L的重组HEL。重组溶菌酶的高产率使我们能够在人体中进行感官分析。纯化的重组溶菌酶与直接从蛋清中纯化的溶菌酶一样，能引发甜味感觉，并对藤黄微球菌细胞显示出完全的裂解活性。这些结果表明，带有杀稻瘟菌素S选择系统的巴斯德毕赤酵母表达系统可用于高产率地生产具有活性形式的重组甜味蛋白。
