Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling

Abstract

Green fluorescent protein (GFP) 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.

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

“…The AAV vectors used in these studies were constructed by inserting either the rat brain-derived neurotrophic factor (BDNF) or 'humanized' green fluorescent protein (GFP) 62 cDNA into the AAV vector plasmid pTR-UF. 63 Briefly, BDNF cDNA was cloned into pcDNA I/AMP yielding the plasmid pc5-BDNF.…”

A fluorescent p53GFP fusion protein facilitates its detection in mammalian cells while retaining the properties of wild-type p53

“…This was accomplished by fusing the wild-type human p53 gene with a highly¯uorescent mutant of the A. victoria green¯uorescent protein (cycle3 GFP) (Crameri et al, 1996). Cycle3 GFP has been optimized for codon usage by eukaryotic cells and contains three mutations which convert hydrophobic residues to hydrophilic ones (F100S, M154T and V164A).…”

“…These changes result in a readily expressed protein with increased solubility in eukaryotic cells. Additionally, the cycle3 mutant-GFP¯uoresces 46-times more brightly than wild-type GFP at 520 nm as determined by FACS analysis of transduced Chinese hamster ovary cells (Crameri et al, 1996).…”

From green to blue: Site‐directed mutagenesis of the green fluorescent protein to teach protein structure–function relationships

“…Ampicillin (60 lg/mL) and tetracycline (30 lL/mL) were used as antibiotics. Plasmid pGFPCR [4] carries the cycle 3 variant of GFP (F99S, M153T, and V163A) described by Crameri et al [3] and is derived from pGFPuv plasmid (Clontech, Mountain View, CA). Either plasmid can be used in this course.…”

“…Although the changes in spectral properties of GFP usually involve the mutation of several residues in the protein [2], making it nonpractical in a laboratory course for undergraduate students, we propose the implementation of only two mutations, Y66H and Y145F in the ''cycle 3'' variant of GFP (F99S, M153T, and V163A) or GFPuv with an excitation maximum at 385 nm [3]. The single Y66H mutation in GFPuv is enough to produce a change in fluorescence emission from green (510 nm) to blue (450 nm) and therefore allows students to correlate the phenotype of the mutated protein with the induced structural changes.…”