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首页> 外文期刊>Applied Microbiology and Biotechnology >Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris
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Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris

机译：巴斯德毕赤酵母补料分批发酵生产重组9和15 kDa颗粒溶素

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摘要

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.
机译：颗粒溶素是一种由人类溶细胞性T淋巴细胞和自然杀伤细胞产生的溶细胞促炎蛋白。颗粒溶素具有两种稳定的同工型，分子量分别为9和15 kDa。 9 kDa形式是15 kDa前体蛋白水解成熟的结果。重组9-kDa颗粒溶素对多种微生物（如细菌，寄生虫，真菌，酵母菌和多种肿瘤细胞系）表现出细胞溶解活性。然而，通过传统方法难以大量生产颗粒溶素。在这项研究中，我们开发了一种简单而强大的补料分批发酵工艺，用于在高细胞密度的基础盐培养基中使用巴斯德毕赤酵母生产和纯化重组9-kDa和15-kDa颗粒溶素。发酵过程中颗粒溶素的产量至少达到100 mg / l，使用常见的His-select亲和力和离子交换色谱法可达到95％以上的纯度。功能分析表明，酵母表达的颗粒溶素显示出剂量依赖性靶细胞毒性。这些结果表明，巴斯德毕赤酵母中的发酵为大规模重组粒状溶素的生产提供了合理的策略，可用于临床应用和基础研究。

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