PDF(Pubmed)
Abstract：
Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 °C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.
摘要：
这里,我们通过多轮DNA和蛋白质工程增强了流行的酵母展示方法。我们介绍了为记者量身定制的表面曝光，eUnaG2和DnbALFA，建立了一个新的C和N末端融合向量平台。eUnaG2的优化导致比UnaG明亮五倍的荧光和10°C的热稳定性增加。优化的DnbALFA具有起始蛋白表达水平的10倍。在此之后,开发了不同的质粒以创建一个复杂的平台，从而允许广泛的蛋白质表达组织和标记策略。与传统的pCTcon2和c-myc标记相比，我们的平台显示出非表达和表达细胞之间的分离效果高达五倍。允许更少的选择轮次并实现更高的结合亲和力。检测16种不同的蛋白质,与c-myc标记相比，增强的系统始终显示出更强的表达信号。除了简单的收益，速度,和成本效益，引入了新的应用来监测蛋白质表面暴露和分泌途径中的蛋白质滞留，从而成功地对难以表达的蛋白质进行蛋白质工程。作为一个例子,我们展示了如何优化ATG16L1蛋白的WD40结构域用于酵母表面和可溶性细菌表达，从不表达蛋白开始。作为第二个例子，我们展示了如何使用这里介绍的增强酵母展示方法，我们快速选择高亲和力结合剂对两个蛋白质靶标，证明了产生新的蛋白质-蛋白质相互作用的简单性。虽然方法上的变化是渐进的，它导致酵母展示对许多应用的适用性的定性增强。
