Comprehensive Screening of a Light-Inducible Split Cre Recombinase with Domain Insertion Profiling.
Comprehensive Screening of a Light-Inducible Split Cre Recombinase with Domain Insertion Profiling.
复制标题
通过域插入分析对光诱导分裂 Cre 重组酶进行全面筛选。
DOI：
10.1021/acssynbio.3c00328
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发表时间：
2023
影响因子：
4.7
通讯作者：
Dunlop,MaryJ
中科院分区：
文献类型：
--
作者：
Tague,Nathan;Andreani,Virgile;Fan,Yunfan;Timp,Winston;Dunlop,MaryJ
用光或化学诱导二聚体裂解蛋白质提供了蛋白质功能翻译后控制的机制。然而，目前用于工程刺激响应性分裂蛋白的方法通常需要大量的蛋白质工程专业知识和对单个构建体的费力筛选。为了应对这一挑战，我们使用混合文库方法，能够并行快速生成和筛选几乎所有可能的分裂蛋白构建体，其中可以通过测序读出结果。我们使用光遗传学二聚体在 Cre 重组酶上执行我们的方法作为概念证明，从而获得整个蛋白质分裂位点的全面数据。为了提高预测分裂蛋白行为的准确性，我们开发了一种贝叶斯计算方法来将实验程序固有的错误置于背景中。总的来说，我们的方法提供了一种简化的方法来实现感兴趣蛋白质的诱导翻译后控制。
Splitting proteins with light- or chemically inducible dimers provides a mechanism for post-translational control of protein function. However, current methods for engineering stimulus-responsive split proteins often require significant protein engineering expertise and the laborious screening of individual constructs. To address this challenge, we use a pooled library approach that enables rapid generation and screening of nearly all possible split protein constructs in parallel, where results can be read out by using sequencing. We perform our method on Cre recombinase with optogenetic dimers as a proof of concept, resulting in comprehensive data on the split sites throughout the protein. To improve the accuracy in predicting split protein behavior, we develop a Bayesian computational approach to contextualize errors inherent to experimental procedures. Overall, our method provides a streamlined approach for achieving inducible post-translational control of a protein of interest.