An HPLC-based Assay to Study the Activity of Cyclic Diadenosine Monophosphate (C-di-AMP) Synthase DisA from Mycobacterium smegmatis

Materials and reagents

Reagents

1. Purified DisA protein (Origin: M. smegmatis)

2. ATP (Sigma, catalog number: A2383)

3. C-di-AMP (Jena Bioscience, catalog number: NU-954S)

4. Tris base (Sigma, catalog number: 10708976001)

5. EDTA (Sigma, catalog number: E4884)

6. Sodium chloride (NaCl) (Sigma, catalog number: S9888)

7. Tetrabutylammonium hydrogen sulfate (Sigma, catalog number: 15583)

8. Methanol (HPLC grade) (Sigma, catalog number: 34860)

9. Double-distilled water (Milli-Q Ultrapure Water Systems)

10. KH₂PO₄ (Sigma, catalog number: P5655)

11. MgCl₂ (Sigma, catalog number: M8266)

12. Tris-Cl (Sigma, catalog number: 10812846001)

13. Luria Bertani Broth, Miller (HIMEDIA, catalog number: M1245)

14. IPTG (Sigma, catalog number I6758)

    
    

1. Dialysis membrane (Sigma, catalog number: D9527)

2. Pipette tips (Tarsons, catalog numbers: 521000, 521010, 521020)

3. 1.5 mL reaction tubes (Tarsons, catalog number: 500010)

4. Membrane filter, 0.22 μm pore size (Merck, catalog number: GSWP04700)

   
   

1. Protein purification buffers: lysis buffer, wash buffer, and elution buffer (see Recipes)

2. DisA dialysis buffer (buffer A) (see Recipes)

3. Size exclusion chromatography (SEC) (buffer B) (see Recipes)

4. Buffer C for c-di-AMP synthesis reaction (see Recipes)

5. Buffer D (see Recipes)

6. Buffer E (see Recipes)

   
   

1. Protein Purification buffers

   Lysis buffer

   50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)

   
   

   Wash buffer

   50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 40 mM imidazole

   
   

   Elution buffer

   50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 300 mM imidazole

   
   

2. DisA dialysis buffer (buffer A)

   50 mM Tris-Cl buffer at pH 7.5, 300 mM NaCl

   
   

3. Size exclusion chromatography (SEC) (buffer B)

   50 mM Tris-Cl (pH 7.9), 300 mM NaCl

   
   

4. Buffer C for c-di-AMP synthesis reaction

   50 mM Tris (pH 9.4), 300 mM NaCl

   
   

5. Buffer D

   100 mM KHPO, 4 mM tetrabutylammonium hydrogen sulfate, pH 5.9

   
   

6. Buffer E

   75% (v/v) buffer D, 25% (v/v) methanol

Equipment

1. Micropipettes with varying capacity (T-2, T-10, T-20, T-100, T-200, T-1000) (Tarsons, catalog numbers: 030000, 030010, 030020, 030030, 030040, 03005)

2. Spectrophotometer (Eppendorf, model: BioSpectrometer^®)

3. HPLC system: Agilent 1200 HPLC (Agilent Technologies, model: Agilent 1200 HPLC) equipped with quaternary pump, autosampler, thermostated column compartment, with a UV detector

4. Äkta (Cytiva, former GE Health care, model: 29707638)

5. C-18 column (4.6 × 150 mm) (Agilent, model: Eclipse XDB-C-18)

6. Superose 12 10/300 Column (Cytiva, catalog number: GE17-5173-01)

7. Dry bath (Bio-Rad, catalog number: 1660563)

8. Refrigerated centrifuge (Eppendorf, model: centrifuge 5430)

9. Vortex shaker (Tarsons, model: SPINIX^TM)

10. Biological safety cabinet (Thermo Scientific, model: 1300 Series Class II, Type A2)

11. Vacuum pump and assembly (Tarsons, model: ROCKYVAC^TM Vacuum Pump with assembly)

12. Water purification system (Millipore, model: Ultra-Pure Water Purification System)

Software and datasets

1. HPLC system software LC/CE Agilent ChemStation (B.04.02 SP1, 4/2010)

2. Graph Pad Prism 5.01 (5.01, 9/2007)

Procedure

1. DisA protein purification and C-di-AMP synthesis assay with DisA protein from Mycobacterium smegmatis

   1. Purify the DisA protein as described in Gautam et al. [12]. The protein purification procedure is briefly described below.

   2. Inoculate E. coli BL21 (DE3) carrying the pET28a plasmid with the disA gene in LB medium and grow overnight at 37 °C. Prepare secondary cultures by inoculating 1% of the primary culture and grow at 37 °C with shaking until the OD reaches 0.6.

   3. Induce the secondary cultures by adding 1 mM isopropyl ß-D-thiogalactopyranoside (IPTG) and incubate for 3 h at 37 °C.

   4. Harvest the cultures by centrifugation at 495× g; then, resuspend the pellet in lysis buffer [50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)].

   5. Sonicate the cells and centrifuge at 1980× g to remove the cell debris.

   6. Load the supernatant onto a Ni-NTA column to allow the protein to bind to the Ni-NTA beads.

   7. Wash the column with 100 column volumes of wash buffer (50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 40 mM imidazole).

   8. Elute the DisA protein using elution buffer containing 50 mM Tris-Cl (pH 7.9), 300 mM NaCl, and 300 mM imidazole. Analyze the protein by 10% SDS-PAGE.

   9. Dialyze the purified DisA protein in Buffer A (see Recipes) for 12–16 h at 4 °C) as per the mentioned protocol [12].

   10. Inject the dialyzed protein into a size exclusion chromatography (SEC) column to further purify the protein against buffer B (see Recipes).

   11. Store the purified protein at -20 °C or -80 °C for future use.

   12. Determine the concentration of the DisA protein (photometric measurement at 280 nm/BCA method).

   13. Calculate the amount of DisA protein required to achieve a concentration of 1 μM in a 50 μL reaction.

   14. In a 1.5 mL Eppendorf tube, set up a 50 μL reaction containing DisA protein (1 μM), 0.5 mM ATP, 5 mM MgCl in Buffer C (see Recipes).

   15. Incubate the reaction at 37 °C for 4 h.

   16. Stop the reaction by adding 10 mM EDTA to the reaction tube.

   17. Centrifuge the reaction sample at 1,530× g for 30 min at 4 °C.

   18. Collect the supernatant for HPLC analysis or store it at -20 °C for future use (Figure 1).

       
       

       
       Figure 1. HPLC profiles of nucleotides and standard curve of c-di-AMP. A. HPLC profile of standard ATP. B. HPLC profile of standard C-di-AMP. C. HPLC profile of C-di-AMP synthesis reaction. D. Standard curve for c-di-AMP.

       
       

2. Generation of c-di-AMP standard curve

   1. Prepare different concentrations of c-di-AMP or ATP (ranging from 100 to 500 μM) stock solutions in buffer C (see Recipes).

   2. Use a reverse phase C-18 column to separate the nucleotides by using Buffer D (see Recipes) and Buffer E (see Recipes).

   3. Filter and degas the HPLC buffers D and E using 0.22 μm filter paper.

   4. Apply the following buffer gradient to separate the nucleotides at a flow rate of 0.7 mL/min:

      0.0 min: 0% buffer E

      2.5 min: 0% buffer E

      5.0 min: 30% buffer E

      10.0 min: 60% buffer E

      14.0 min: 100% buffer E

      21.0 min: 100% buffer E

      22.0 min: 50% buffer E

      23.0 min: 0% buffer E

   5. Inject 20 μL of 500 μM pure c-di-AMP and then ATP separately for HPLC analysis.

   6. After each sample injection, wash the HPLC column with buffer D for 10 min at a flow rate of 0.7 mL/min.

   7. Pure c-di-AMP should show a peak at ~23.7 min, and ATP should show a peak at ~17.4 min.

   8. After this preliminary validation, inject 20 μL of different concentrations (100–500 μM) of c-di-AMP into the HPLC and analyze using the previously stated gradient and buffers.

   9. Use the chromatography data system software to obtain the peak area.

   10. Prepare the standard curve by plotting the peak area under the curve (AUC) vs. the concentration of c-di-AMP used.

       
       

3. Analysis of the C-di-AMP synthesis assay reaction by HPLC

   1. Use 20 μL of reaction sample for HPLC analysis.

   2. Inject the reaction sample into the HPLC system using the gradient described above.

   3. Observe for a peak at approximately ~23.7 min.

   4. If a peak is observed at ~23.7 min, determine the area under the peak using the chromatography data system software.

   5. Use the peak area to apply the standard curve and linear equation to determine the concentration (μM) of c-di-AMP formed in the reaction.

   6. Collect the HPLC elution fractions around 23.7 min to further confirm the mass of the reaction product (See General note 2).

General notes and troubleshooting

1. The synthesis of c-di-AMP by DisA (a DAC domain-containing protein) highly depends on assay conditions like buffer compositions (NaCl/KCl), pH (5.4–9.4), temperature (35–40 °C), and incubation time (0–6 h). For Mycobacterium smegmatis, the highest activity was observed in Buffer C (50 mM Tris, 300 mM NaCl, pH 9.4). After 4 h of incubation at 37 °C, complete utilization of ATP was noted. When using DisA from different sources, activity optimization might be necessary to avoid multiple peaks (e.g., ATP or intermediate products).

2. We employ an earlier reported LC-MS method to analyze the HPLC eluted fractions from the DisA reactions (Burker Daltonics, Germany) as described by [14]. MS–MS analysis (negative or positive ion mode) of the eluted molecular masses is performed to verify the presence of c-di-AMP. The standard protocol involves LC–MS and MS–MS analysis using the HPLC gradient composition, as previously reported [15].

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

1. Ryjenkov DA et al. [10]. Cyclic diguanylate is a ubiquitous signalling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J Bacteriol. [Fig 2B, Fig 3B,C, Used for c-di-GMP analysis].

2. Christen M et al. [11]. Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J Biol Chem. [Fig 2A, Used for c-di-GMP analysis].

1. Bai Y et al. [13]. Mycobacterium tuberculosis Rv3586 (DacA) is a diadenylate cyclase that converts ATP or ADP into c-di-AMP. PLoS One. [Fig 2A, Used for C-di-AMP analysis]

Acknowledgments

D.C. gratefully acknowledges the initial financial help through a grant from the Dept. of Biotechnology, Govt. Of India, through a grant number DBT/PR33123/MED/29/1497/2020.

Competing interests

The authors declare no competing interests.

References

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