Lanser Dylan M, Gelli Angie
Department of Pharmacology, School of Medicine, University of California, Davis, CA, USA 95616.
Bio Protoc. 2023 Mar 5;13(5):e4628. doi: 10.21769/BioProtoc.4628.
Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The yeast is a highly useful eukaryotic protein expression system. 's low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract.
高质量的重组蛋白是所有下游应用的关键起始材料，但蛋白质固有的复杂性及其表达和纯化过程带来了巨大挑战。酵母是一种非常有用的真核蛋白表达系统。其低成本、遗传易操作性、快速的基因表达以及可扩展性使其成为外源蛋白的理想表达系统。在此，我们开发了一种方案，该方案优化了一种非哺乳动物分泌型金属蛋白酶的表达和分离，通过该方案我们能够常规地生成纯净且具有蛋白水解活性的重组蛋白。我们通过改变补料方式，即在甲醇诱导阶段采用不可发酵且无抑制作用的碳源，实现了生长和蛋白质产量的最大化。这种方法提高了生物量产量，并产生了毫克级的重组蛋白。这种改进的标准化方法极大地促进了涉及活性重组真菌蛋白酶的下游应用，如与纳米颗粒的偶联以及与结构相关的研究。图形摘要。
