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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Published: March 19th, 2021

DOI:

10.3791/62331

1Department of Biology, University of Copenhagen

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Bacteria use several small signaling molecules to adapt to constantly changing environments1,2. For example, the autoinducers, N-acylhomoserine lactones and their modified oligopeptides, mediate the intercellular communication among bacteria to coordinate population behavior, a phenomenon known as quorum sensing2. Another group of small signaling molecules is the NSMs, including the widely studied cyclic adenosine monophosphate (cAMP), cyclic di-AMP, cyclic di-guanosine monophosphate (cyclic di-GMP), and guanosine penta- and tetra phosphates (p)ppGpp1. B....

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1. Preparation of whole cell lysates

  1. Inoculate the E. coli K-12 ASKA ORFeome collection strains15 into 1.5 mL Lysogeny broth (LB) containing 25 µg/mL chloramphenicol in 96-well deep well plates. Grow overnight (O/N) for 18 h at 30 °C with shaking at 160 rpm. On the next day, add isopropyl β-d-1-thiogalactopyranoside (IPTG) (final 0.5 mM) to the O/N cultures to induce protein expression at 30 °C for 6 h.
  2. Pellet cells at 500 x g for 10 min........

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Following the above-described protocol will typically yield two types of results (Figure 3).

Figure 3A shows a plate with relatively low background binding signals (binding fractions < 0.025) from the majority of wells. The positive binding signal from the well H3 gives a binding fraction of ~0.35 that is much higher than that observed for the other wells. Even without quantification, well H3 is remarkable, suggesting that a targe.......

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One of the critical steps in performing DRaCALA screening is to obtain good whole cell lysates. First, the tested proteins should be produced in large amounts and in soluble forms. Second, the lysis of cells should be complete, and the viscosity of the lysate must be minimal. The inclusion of lysozyme and the use of three cycles of freeze-thaw are often enough to lyse cells completely. However, the released chromosomal DNA makes the lysate viscous and generates high background binding signal, resulting in false positives.......

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The work is supported by an NNF Project Grant (NNF19OC0058331) to YEZ, and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (Nº 801199) to MLS.

Equation 1

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Name Company Catalog Number Comments
32P-α-GTP Perkinelmer BLU006X250UC
96 x pin tool V&P Scientific VP 404 96 Bolt Replicator, on 9 mm centers, 4.2 mm Bolt Diameter, 24 mm long
96-well V-bottom microtiter plate Sterilin MIC9004 Sterilin Microplate V Well 611V96
Agar OXOID - Thermo Fisher LP0011 Agar no. 1
ASKA collection strain NBRP, SHIGEN, JAPAN Ref: DNA Research, Volume 12, Issue 5, 2005, Pages 291–299. https://doi.org/10.1093/dnares/dsi012
Benzonase SIGMA E1014-25KU genetically engineered endonuclease from Serratia marcescens
Bradford Protein Assay Dye Bio-Rad 5000006 Reagent Concentrate
DMSO SIGMA D8418 ≥99.9%
DNase 1 SIGMA DN25-1G
gel filtration10x300 column GE Healthcare 28990944 contains 20% ethanol as preservative
Glycerol PanReac AppliChem 122329.1214 Glycerol 87% for analysis
Hypercassette Amersham RPN 11647 20 x 40 cm
Imidazole SIGMA 56750 puriss. p.a., ≥ 99.5% (GC)
IP Storage Phosphor Screen FUJIFILM 28956474 BAS-MS 2040 20x 40 cm
Isopropyl β-d-1-thiogalactopyranoside (IPTG) SIGMA I6758 Isopropyl β-D-thiogalactoside
Lysogeny Broth (LB) Invitrogen - Thermo Fisher 12795027 Miller's LB Broth Base
Lysozyme SIGMA L4949 from chicken egg white; BioUltra, lyophilized powder, ≥98%
MgCl2 (Magnesium chloride) SIGMA 208337
MilliQ water ultrapure water
multichannel pipette Thermo Scientific 4661110 F1 - Clip Tip; 1-10 ul, 8 x channels
NaCl VWR Chemicals 27810 AnalaR NORMAPUR, ACS, Reag. Ph. Eur.
Ni-NTA Agarose Qiagen 30230
Nitrocellulose Blotting Membrane Amersham Protran 10600003 Premium 0.45 um 300 mm x 4 m
PBS OXOID - Thermo Fisher BR0014G Phosphate buffered saline (Dulbecco A), Tablets
PEG3350 (Polyethylene glycol 3350) SIGMA 202444
phenylmethylsulfonyl fluoride (PMSF) SIGMA 93482 Phenylmethanesulfonyl fluoride solution - 0.1 M in ethanol (T)
Phosphor-imager GE Healthcare 28955809 Typhoon FLA-7000 Phosphor-imager
Pipette Tips, filtered Thermo Scientific 94410040 ClipTip 12.5 μl nonsterile
Poly-Prep Chromatography column Bio-Rad 7311550 polypropylene chromatography column
Protease inhibitor Mini Pierce A32955 Tablets, EDTA-free
screw cap tube Thermo Scientific 3488 Microcentifuge Tubes, 2.0 ml with screw cap, nonsterile
SLS 96-deep Well plates Greiner 780285 MASTERBLOCK, 2 ML, PP, V-Bottom, Natural
spin column Millipore UFC500396 Amicon Ultra -0.5 ml Centrifugal Filters
Thermomixer Eppendorf 5382000015 Thermomixer C
TLC plate (PEI-cellulose F TLC plates) Merck Millipore 105579 DC PEI-cellulose F (20 x 20 cm)
Tris SIGMA BP152 Tris Base for Molecular Biology
Tween 20 SIGMA P1379 viscous non-ionic detergent
β-mercaptoethanol SIGMA M3148 99% (GC/titration)

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