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

Summary

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.

Abstract

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.

Introduction

Bacteria use several small signaling molecules to adapt to constantly changing environments^1,2. 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....

Protocol

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........

Discussion

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.......

Materials

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)