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| | Fluorescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of GFP Chromophore Formation |
| Description: | Keywords: GFP, refolding, reducing reagent, GFP fluorescence Assay Overview: M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. In primary screenning, the RecA intein splicing activity was followed by GFP fluorescence. The inhibition of GFP fluorescence could arise either from inhibition from the inhibition of protein splicing or from the inhibition of GFP chromophore formation. In this counter screen, cpds were tested with denatured GFP protein purified as the 4-thiopyridine derivative in 8 M urea to rule out false positives which interupt GFP chromophore formation. Specifically, 4-thiopyridine modified GFP was incubated with compounds first in 1536 well plates. the GFP chromophore formation was initiated by addition of |
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