![]() coli expressing each of the seven generations of cp193 generated during the directed evolution process. 21 (B) Fluorescence image of a streak plate of E. (A) Representation of the steps of modification that produced the original cp193 variant from the insertional variant i193. The relative steadfastness of the intrinsic fluorescent properties of the FP allow researchers to confidently assume that fluorescent intensity is directly proportional to protein concentration in live cell imaging experiments.Įngineering of a bright circularly permuted mCherry variant. As the chromophore is isolated, its inherent fluorescent properties (e.g., quantum yield, extinction coefficient, lifetime, and rate of photobleaching) are presumably less susceptible to changes in the protein environment (e.g., changes in pH, viscosity, and concentration of quenchers of fluorescence) than if the chromophore was exposed to the bulk solvent. 1 Accordingly, expression of a FP gene chimera in a wide variety of cell-types and tissue-types can provide a researcher with a noninvasive fluorescent marker for imaging of fusion protein localization and dynamics.įor many applications of FPs, the fact that the FP chromophore is fully encapsulated inside an 11-stranded ‘β-can’ tertiary structure, 2 and thus effectively isolated from the environment of the bulk solvent, is advantageous. ![]() Their usefulness stems from the fact that these proteins are self-sufficient to form a brightly fluorescent chromophore within the confines of their own tertiary structure. These new variants may ultimately prove useful for the creation of single FP-based Ca 2+ biosensors.Ī variety of intrinsically fluorescent proteins (FP) from the superfamily of Aequorea victoria green FP (avGFP)-like proteins have proven to be popular and powerful tools for live cell fluorescence imaging applications. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β-strand of mCherry. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole–dipole orientation. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca 2+ and other analytes of interest. ![]() Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. ![]()
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