PMID:12614622
| Citation |
Philipps, B, Hennecke, J and Glockshuber, R (2003) FRET-based in vivo screening for protein folding and increased protein stability. J. Mol. Biol. 327:239-49 |
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| Abstract |
Fluorescence resonance energy transfer (FRET) was used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the cytoplasm of Escherichia coli. The system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue-fluorescent protein (BFP) to the N terminus of protein X. Efficient FRET from BFP to GFP in the ternary fusion protein is observed in vivo only when protein X is folded and brings BFP and GFP into close proximity, while FRET is lost when BFP and GFP are far apart due to unfolding or intracellular degradation of protein X. The screening system was validated by identification of antibody V(L) intradomains with increased thermodynamic stabilities from expression libraries after random mutagenesis, bacterial cell sorting, and colony screening. |
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| Keywords |
Amino Acid Sequence; Antibodies/chemistry; Antibodies/immunology; Base Sequence; Blotting, Western; Cytoplasm/metabolism; Escherichia coli/cytology; Escherichia coli/genetics; Escherichia coli/metabolism; Fluorescence Resonance Energy Transfer/methods; Green Fluorescent Proteins; Luminescent Proteins/analysis; Molecular Sequence Data; Protein Folding; Protein Structure, Tertiary; Recombinant Fusion Proteins/analysis; Recombinant Fusion Proteins/chemistry; Reproducibility of Results; Thermodynamics |
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