A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets.
A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets.
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DOI：
10.1021/acssynbio.1c00395
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发表时间：
2021-12-17
影响因子：
4.7
通讯作者：
Schreiber G
中科院分区：
文献类型：
--
作者：
Zahradník J;Dey D;Marciano S;Kolářová L;Charendoff CI;Subtil A;Schreiber G
在这里，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。我们引入了表面修饰的报告基因eUnaG 2和DnbALFA，创建了一个新的C端和N端融合载体平台。eUnaG 2的优化导致比UnaG亮五倍的荧光和增加10 °C的热稳定性。优化的DnbALFA的表达水平是起始蛋白的10倍。在此之后，开发了不同的质粒以创建一个复杂的平台，允许广泛的蛋白质表达组织和标记策略。与传统的pCTcon 2和c-myc标记相比，我们的平台在非表达和表达细胞之间的分离效果好五倍，允许更少轮的选择并实现更高的结合亲和力。测试了16种不同的蛋白质，增强的系统显示出比c-myc标记更强的表达信号。除了简单性、速度和成本效益方面的收益外，还引入了新的应用程序来监测蛋白质表面暴露和分泌途径中的蛋白质保留，从而成功地对难以表达的蛋白质进行蛋白质工程改造。作为一个例子，我们展示了我们如何优化的WG 40域的ATG 16 L1蛋白酵母表面和可溶性细菌表达，从非表达蛋白质。作为第二个例子，我们展示了如何使用这里提出的增强酵母展示方法，我们迅速选择高亲和力的粘合剂对两个蛋白质的目标，证明了简单的产生新的蛋白质-蛋白质相互作用。虽然方法上的变化是渐进的，但它导致酵母展示在许多应用中的适用性得到了质的提高。
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.
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影响因子：
6.8
作者：
Gai, S. Annie;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane
影响因子：
5.7
作者：
Cohen-Khait, Ruth;Dym, Orly;Schreiber, Gideon
通讯作者：
Schreiber, Gideon
影响因子：
14.8
作者：
Drew, David;Newstead, Simon;Iwata, So
通讯作者：
Iwata, So
影响因子：
4.4
作者：
Eiden-Plach, A;Zagorc, T;Schmitt, MJ
通讯作者：
Schmitt, MJ
DOI：
10.1073/pnas.1613122113
发表时间：
2016-12-27
影响因子：
11.1
作者：
Cohen-Khait, Ruth;Schreiber, Gideon
通讯作者：
Schreiber, Gideon