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

Applied Biochemistry and Biotechnology
Xueying WangZongbao K Zhao

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...Continue Reading

References

Jul 15, 1999·Nature Biotechnology·G S WaldoT C Terwilliger
Jun 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Robert E CampbellRoger Y Tsien
Dec 4, 2002·Nature Reviews. Molecular Cell Biology·Jin ZhangRoger Y Tsien
Jan 28, 2003·Current Opinion in Chemical Biology·Geoffrey S Waldo
Nov 22, 2005·Nature Methods·Nathan C ShanerRoger Y Tsien
Dec 22, 2005·Nature Biotechnology·Jean-Denis PédelacqGeoffrey S Waldo
Feb 17, 2006·Journal of Biochemical and Biophysical Methods·Fusinita van den Ent, Jan Löwe
Apr 6, 2006·Journal of the American Chemical Society·Liping ZhangRebekka M Wachter
Jan 12, 2007·Protein Expression and Purification·Jinxia WangZongbao Kent Zhao
Jun 23, 2007·Protein Engineering, Design & Selection : PEDS·Catherine Heddle, Sabine L Mazaleyrat
Oct 16, 2007·Journal of Microbiological Methods·Jinxia WangZongbao Kent Zhao
Apr 17, 2008·Applied Microbiology and Biotechnology·Ping Lu, Ming-Guang Feng
Oct 22, 2008·Nature Methods·Rinkoo D Gupta, Dan S Tawfik
Nov 29, 2008·Journal of Structural and Functional Genomics·Pawel ListwanGeoffrey S Waldo
Sep 2, 2009·Current Opinion in Biotechnology·Michael J Dougherty, Frances H Arnold
Feb 23, 2010·Analytica Chimica Acta·Pu ChenBi-Feng Liu

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