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Analysis of Fluorescent Proteins for the Development of an In Vivo System for Protease Evolution
Author(s) -
Selby Thomas Lee
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.604.2
Subject(s) - förster resonance energy transfer , green fluorescent protein , proteolysis , fusion protein , biophysics , protease , fluorescence , chromophore , chemistry , in vivo , yellow fluorescent protein , biochemistry , recombinant dna , biology , photochemistry , enzyme , gene , physics , quantum mechanics , microbiology and biotechnology
The objective of this study is to develop a robust in vivo system using E. coli cells for screening protease activity based on intracellular Förster (fluorescence) resonance energy transfer (FRET). Toward this end, we expressed and purified the fluorescent proteins GFP, Orange‐FP (OFP), Clover, Ruby2, and td‐Tomato as single polypeptides, and as fusion partners linked by protease target sequences, to evaluate their stability, fluorescence intensity, chromophore maturation, kinetics, and FRET properties under various expression and reaction conditions. GFP and Clover showed similar spectroscopic properties when produced by constant induction, or mid‐log phase induction. However, the spectroscopic properties of OFP, Ruby2, and td‐Tomato varied significantly based on their induction conditions, cell density, and growth media. These differences appear to be related to the degree of maturation of the chromophore, which required optimization to achieve maximum FRET, but resulted in aggregation for some of the proteins under certain conditions. The rate of chromophore maturation in vitro varied depending on conditions and/or the presence of a fusion partner. In fusion constructs, the fully matured, fluorescent proteins demonstrated a constant decrease in FRET during proteolysis of the target sequence. However, interpretation of the kinetic profiles in vitro was complicated by samples with incomplete maturation. This complication is believed to be due a simultaneous decrease in FRET signal upon proteolysis, as well as an increase in FRET occurring during chromophore maturation. While the Clover‐Ruby2 fusion protein demonstrated a FRET efficiency that was superior to the GFP‐OFP protein, the hydrophobic Ruby2 protein was prone to aggregation under certain reaction conditions, resulting in a loss of fluorescent signal, challenges during purification, and difficulty interpreting kinetic results. Td‐Tomato, a less hydrophobic protein, gave lower yields during purification, and matured in vivo at a much slower rate than Ruby2 and OFP. In conclusion, these results revealed variable fluorescent properties for different fusion partners investigated in vitro , while providing data for comparison when optimizing fluorescent protein expression, and kinetics, during in vivo screening, with the ultimate goal of developing a system that will utilize fluorescent activated cell sorting (FACS) to evolve proteases with novel specificities. Support or Funding Information This work was supported by the National Institutes of Health under grant GM080691 and Mercer University School of Medicine, Division of Basic Medical Sciences.