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Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution

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Abstract

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 the nadD gene, used to transform Escherichia coli DH10B 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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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers: 21325627, 21572227) and Dalian Institute of Chemical Physics, CAS (grant number: DICP ZZBS201605).

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Authors and Affiliations

  1. Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, People’s Republic of China

    Xueying Wang, Lei Wang, Xinping Lin, Xiaobing Yang, Wujun Liu & Zongbao K. Zhao

  2. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Xueying Wang

  3. Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, People’s Republic of China

    Zongbao K. Zhao

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  1. Xueying Wang
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  2. Lei Wang
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  3. Xinping Lin
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  4. Xiaobing Yang
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  5. Wujun Liu
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  6. Zongbao K. Zhao
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Correspondence to Zongbao K. Zhao.

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Wang, X., Wang, L., Lin, X. et al. Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution. Appl Biochem Biotechnol 185, 81–90 (2018). https://doi.org/10.1007/s12010-017-2640-z

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  • DOI: https://doi.org/10.1007/s12010-017-2640-z

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