Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins

1. Preparation of whole cell lysates

1. Inoculate the E. coli K-12 ASKA ORFeome collection strains 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. Freeze the pellets at -80 °C until use. To lyse the cells, add 150 µL of lysis buffer L1 (40 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl₂, supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 40 µg/mL DNase 1, and 0.5 mg/mL lysozyme) to resuspend the pellet.
3. Freeze the cells at -80 °C for 30 min, and then thaw at 37 °C for 20 min. Repeat this cycle three times to lyse the cells. Store the lysates at -80 °C before use.

2. Purification of Rel_seq and GppA

NOTE: The recombinant proteins Rel_seq from Streptococcus equisimilis and GppA from E. coli K-12 are used to synthesize the radiolabeled pppGpp and ppGpp, respectively.

1. Grow and collect cells overexpressing each protein.
   1. Grow the E. coli BL21 DE3 strain up to exponential phase (optical density (OD) ~0.3-0.4) in LB broth, and spin down 1 mL of culture at 6000 x g for 5 min. Decant the supernatant, and resuspend the cells with 100 µL of ice-cold TSB broth (LB broth supplemented with 0.1 g/mL PEG3350, 0.05 mL/mL dimethylsulfoxide, 20 mM MgCl₂).
   2. Mix the plasmids bearing the histidine-tagged relseq and gppA, each 100 ng, with the above cell suspensions in TSB, and incubate on ice for 30 min. Heat-shock the cells at 42 °C for 40 s. Place the mixture on ice for 2 min, and add 1 mL of LB broth at room temperature to allow the cells to recover for 1 h at 37 °C with agitation at 160 rpm.
   3. Plate the recovered cells on LB agar plates supplemented with the corresponding antibiotics (Rel_seq:100 µg/mL ampicillin; GppA: 30 µg/mL kanamycin).On the next day, inoculate the colonies in LB broth to start O/N precultures of both strains at 37 °C.
   4. After 18 h, inoculate 500 mL of LB medium with 10 mL of the O/N cultures and the corresponding antibiotics. Grow the cultures by shaking at 160 rpm at 37 °C. When the OD_600nm reaches 0.5-0.7, induce protein expression by adding 0.5 mM IPTG and growing for 3 h at 30 °C with shaking at 160 rpm.
   5. Collect the cells by spinning at 6084 x g for 10 min at 4 °C. Resuspend the pellet in 20 mL of ice-cold 1x phosphate-buffered saline (PBS), and re-centrifuge at 1912 x g for 20 min at 4 °C. Decant the supernatant, and freeze the pellets at -20 °C before use.
2. Nickel-nitrilotriacetic acid (Ni-NTA) affinity purification
   NOTE: From this point onwards, ensure that the samples are cold.
   1. Add 40 mL of ice-cold lysis buffer L2 (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole, 10 mM β-mercaptoethanol supplemented with protease inhibitors (EDTA-free tablet; see the Table of Materials) to resuspend the pellet. Lyse the cells via sonication (60% amplitude, 2 s ON/ 4 s OFF for 8 min ON). Clear the lysate by spinning at 23,426 x g for 40 min at 4 °C, and continue with the supernatant for purification.
   2. During the above centrifugation, prepare the Ni-NTA resin.
      1. Transfer 500 µL of homogenized Ni-NTA resin into a standing polypropylene chromatography column, and let it settle for 15 min and the storage solution drain through. Wash the resin with 15 mL of ultrapure water twice, and then wash the column with 15 mL of the lysis buffer L2.
   3. Load the cleared supernatant of cell lysate from step 1 onto the column, and let it flow through. Wash the column with 30 mL of washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 20 mM imidazole).
   4. Elute the proteins with 400 µL of the elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 500 mM imidazole) three times. Then, repeat elution with another 300 µL of the elution buffer. Combine the eluted proteins to a final volume of 700 µL.
3. Gel filtration
   1. Prepare gel filtration buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5% glycerol). Wash the size exclusion column with one column volume (25 mL) of the gel filtration buffer.
   2. Load the above 700 µL sample by using a 500 µL loop, run at 0.5 mL/min, and collect 2-3 fractions, each of 0.5 mL volume, containing the respective proteins.
   3. Combine and concentrate the fractions containing each of the proteins using a spin column, and measure the protein concentration using the Bradford assay.

3. Synthesis of ³²P-labeled pppGpp and ppGpp

1. Assemble a small-scale Rel_seq reaction in a screw cap tube (see Table 1).
   NOTE: Work with radioactive reagents only in a licensed place and with personal protective equipment.
2. Incubate the tube at 37 °C in a thermomixer for 1 h, then at 95 °C for 5 min, and place on ice for 5 min. Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized ³²P-pppGpp) to a new screw cap tube.
3. To synthesize ³²P-ppGpp from ³²P-pppGpp, transfer half of the ³²P-pppGpp product to a new screw cap tube, and add 1 µM GppA. Incubate the tube at 37 °C for 10 min, at 95 °C for 5 min, and then place on ice for 5 min.
4. Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized ³²P-ppGpp) to a new screw cap tube.
5. Analyze the ³²P-pppGpp and ³²P-ppGpp by running 1 µL of the samples on a thin layer chromatography (TLC) plate (polyethyleneimine-modified cellulose TLC plates) using the 1.5 M KH₂PO₄, pH 3.4, as mobile phase.
   NOTE: Use the α-³²P-labeled guanosine 5'-triphosphate (³²P-α-GTP) as control.
6. Dry the TLC plate completely, place it between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize and quantify the signals by using a phosphorimager.
   NOTE: When the ratios of  ³²P-pppGpp and  ³²P-ppGpp are higher than 85%, a large-scale reaction (500 µL, sufficient for screening 20 96-well plates) could be assembled and synthesized by using Table 1.

4. DRaCALA screening of the target proteins of (p)ppGpp

1. Thaw and transfer 20 µL of whole cell lysates to a 96-well V-bottom microtiter plate. Add 2.5 U/well of the endonuclease from Serratia marcescens, and incubate at 37 °C for 15 min to reduce lysate viscosity. Place the lysates on ice for 20 min.
2. Mix the  ³²P-pppGpp and  ³²P-ppGpp in a 1:1 ratio, and add 1x lysis buffer L1 to make the final concentration of (p)ppGpp equal to 4 nM.
   NOTE: Given the chemical similarity between pppGpp and ppGpp, a mix of both chemicals will simplify the screening process.
3. Use a multichannel pipette and filtered pipette tips to add and mix 10 µL of the (p)ppGpp mixture with the cell lysate. Incubate at room temperature (RT) for 5 min.
4. Wash the 96 x pin tool by placing it in a 0.01% solution of non-ionic detergent for 30 s, and dry it on tissue paper for 30 s. Repeat the washing of the pin tool 3x.
5. Place the pin tool in the above 96-well sample plates, and wait for 30 s. Lift the pin tool straight up, and place it straight down on a nitrocellulose membrane for 30 s.
   NOTE: If a spot is missing, spot 2 µL of the corresponding samples with a pipette and filtered tips. It is advisable to make a duplicate spot of the same sample as indicated below.
6. Dry the membrane for 5 min at RT. Place the membrane between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize by using a phosphorimager.

5. Quantification and identification of potential target proteins

1. Use the analysis software associated with the phosphorimager to open the .gel file of the visualized plates. Use the Array analysis function to define the 96 spots by setting up a grid of 12 columns x 8 rows.
2. Define big circles to circumscribe the outer edge of the whole spots (see Figure 1). Export the Volumn+Background and Area of the defined big circles, and save in a spreadsheet.
   NOTE: If required, reposition each individual circle to perfectly overlap with the spots, and resize each individual circle to make it slightly bigger than the actual spot.
3. Size down the defined circles to circumscribe the small inner dots. Export the Volumn+Background, and Area of the defined small circles, and save in a spreadsheet.
4. Calculate the binding fractions in the spreadsheet by using the equation in Figure 1, and plot the data. Identify the potential binding proteins in the wells that show high binding fractions in comparison to the majority of other wells.

Table 1: Assembling information for the small- and large-scale synthesis reactions of  ³²P-labeled pppGpp. *10x Relseq buffer contains 250 mM Tris-HCl, pH 8.6; 1M NaCl; 80 mM MgCl₂. Abbreviation: pppGpp = guanosine pentaphosphate.