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Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling

Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling Green fluorescent protein (QFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling

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References (20)

Publisher
Springer Journals
Copyright
Copyright © 1996 by Nature Publishing Company
Subject
Life Sciences; Life Sciences, general; Biotechnology; Biomedicine, general; Agriculture; Biomedical Engineering/Biotechnology; Bioinformatics
ISSN
1087-0156
eISSN
1546-1696
DOI
10.1038/nbt0396-315
Publisher site
See Article on Publisher Site

Abstract

Green fluorescent protein (QFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Journal

Nature Biotechnology – Springer Journals

Published: Mar 1, 1996

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