Fluorescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of RecA-Intein Splicing Activity
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. For primary screening, RecA intein fusion protein was incubated with compounds more ..
BioActive Compounds: 1875
Keywords: GFP-RecA intein, protein splicing, refolding, reducing reagent, GFP fluorescence
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. For primary screening, RecA intein fusion protein was incubated with compounds first in 1536 well plates. The RecA intein splicing activity was initiated by addition of reducing reagent TCEP. GFP fluorescence, produced as a result of protein splicing, was read with fluorescence plate reader.
Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to inhibition of RecA-Intein splicing activity.
GFP-RecA Splicing Assay in Dose
1. Dispense positive control
Dispense 100 nL of positive control ZnCl2(60mM) using Combi nl in respective wells according to plate design to 1536-well assay ready plates (Aurora 00027830) that contain 22.5 nL/well of 10 mM compound in 8 doses with 3-fold dilution(generated by Echo acoustic dispenser)
2. Dispense GFP-RecA Intein
Add 3 uL/well of GFP-recA intein(2uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes.
3. Dispense TCEP to initiate the reaction
Add 3uL/well of TCEP(2mM) with Combi nL (Thermo), incubate at room temperature for ~18 hours
Centrifuge the plates at 1000 rpm x1min
5. Read on Envision for Fluorescence(Ex.405nm/Em.510nm)
20 mM NaPi 7.0
500 mM NaCl
500 mM L-Arginine.HCL
0.01 % Tritin-100
1 % DMSO
ZnCl2 (in H2O, Sigma 229997-50G)
60 mM ZnCl2
TCEP (in intein buffer, Sigma 646547)
2 mM TCEP
GFP-RecA intein (in intein buffer, provided by Henry Paulus's lab)
2 uM GFP-RecA Intein
Dose Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.
Normalization and Pattern Correction
The raw signal was normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of -100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.
No plate pattern correction algorithm method was applied.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(IC50)
Activity_Outcome = 1 (inactive) when
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
IC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
Activity_Outcome = 4 (unspecified) when
no usable data was obtained from the tested compound
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)