Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution
Xueying Wang1,2, Lei Wang1, Xinping Lin1
1Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, People's Republic of China.
Applied Biochemistry and Biotechnology
|October 31, 2017
Summary
This study presents a new method using monomeric red fluorescent protein (mRFP) to easily identify soluble protein mutants in directed evolution. This fluorescent tag helps reduce screening costs and workload by visualizing protein solubility.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Directed evolution generates large protein mutant libraries, often including insoluble variants.
- Identifying and removing insoluble mutants early is essential to reduce screening costs and workload.
Purpose of the Study:
- To develop and demonstrate a method for visualizing soluble protein mutants using a fluorescent fusion tag.
- To improve the efficiency of library screening in directed evolution.
Main Methods:
- A fusion protein construct was created expressing nicotinic acid mononucleotide adenylyltransferase (NadD) with a C-terminal monomeric red fluorescent protein (mRFP) tag.
- Site-saturation mutagenesis was performed on the nadD gene, and transformed E. coli colonies were screened for red fluorescence.
- Fluorescence intensity was correlated with the amount of soluble NadD-mRFP in the cell supernatant.
Main Results:
- A positive correlation was observed between cell culture fluorescence intensity and soluble NadD-mRFP content.
- Mutations at position 132 of NadD resulted in a loss of red fluorescence, indicating a critical role in protein folding.
- The mRFP tag successfully facilitated the identification of soluble mutants for other enzymes, including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase.
Conclusions:
- Monomeric red fluorescent protein (mRFP) serves as an effective fusion reporter for visualizing soluble protein mutants.
- This mRFP-based method enhances the efficiency of library screening in directed evolution by enabling rapid identification of soluble variants.
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