Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution
Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution
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使用单体红色荧光蛋白作为定向进化的报告基因来可视化可溶性蛋白突变体
DOI：
10.1007/s12010-017-2640-z
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发表时间：
2018-05
影响因子：
3
通讯作者：
Zhao Zongbao K.
中科院分区：
文献类型：
--
作者：
Wang Xueying;Wang Lei;Lin Xinping;Yang Xiaobing;Liu Wujun;Zhao Zongbao K.
基于定向进化的蛋白质工程通常会产生大量的含有不溶性突变体的文库，因为突变会导致结构紊乱。为了减少工作量和成本，在专门分析之前识别和消除那些不溶性变体至关重要。在这里，我们展示了一种方法，通过使用单体红色荧光蛋白（mRFP）作为融合标签可视化可溶性蛋白质突变体。设计质粒以表达在C末端与GGGS连接的mRFP标签融合的烟酸单核苷酸腺苷酰转移酶（NadD）。将质粒在thenadD基因内进行位点饱和诱变，用于转化大肠杆菌DH 10 B感受态细胞，导致具有不同红色强度的菌落。发现细胞培养物的荧光强度与上清液中NadD-mRFP突变体的含量呈正相关。在位置132处的突变导致其中大多数菌落失去红色表型的文库，表明该位置对于正确的蛋白质折叠具有关键作用。类似地，mRFP能够鉴定其他酶的可溶性突变体，包括1-脱氧-D-木酮糖-5-磷酸还原异构酶和亚磷酸脱氢酶。这些数据表明，mRFP可以作为一个融合报告可视化可溶性蛋白质突变体，以促进更有效的文库筛选定向进化。
Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within thenadDgene, used to transformEscherichia coliDH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution.
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影响因子：
1.6
作者：
Jinxia Wang;Haidong Tan;Z. Zhao
通讯作者：
Jinxia Wang;Haidong Tan;Z. Zhao
影响因子：
12.9
作者：
Wang, Lei;Ji, Debin;Zhao, Zongbao K.
通讯作者：
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影响因子：
46.9
作者：
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通讯作者：
Waldo, GS
影响因子：
2.2
作者：
Wang, Jinxia;Zhang, Sufang;Zhao, Zongbao (Kent)
通讯作者：
Zhao, Zongbao (Kent)
影响因子：
3.8
作者：
Chao Zhong;P. Wei;Y. Zhang
通讯作者：
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