Parashar Lab

Production of c-di-AMP

Expression and Purification

The sequenced recombinant plasmid containing btDisA was transformed into Escherichia coli BL21(DE3)

competent cells to obtain the btDisA expression strain HJ080.

For protein expression, a single colony of HJ080 was grown overnight at 37 ? C in
Luria-Bertani medium containing 50 ug/mL kanamycin. The overnight culture was
used to inoculate 1000 mL of Luria-Bertani medium (1:100 dilution) supplemented
with 50 ug/mL kanamycin and was grown at 37 ? C to an OD600 of 0.8. After the
addition of IPTG (final concentration of 0.5 mM), the culture was incubated in an
orbital shaker at 28? C for 4 h. The C-His-tagged recombinant protein was purified
using a Ni-NTA resin and eluting in an imidazole concentration of 20, 50, 100, and 250 mM,

respectively. Collected fractions were dialyzed against 20 mM Tris-Cl, pH 8.0, containing 1 mM DTT ,

concentrated in the Bradford assay using BSA as a standard, and then stored in −70 °C.
The purity of the target protein was determined by 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of
the protein was measured by the Bradford method using bovine serum albumin
as a standard.

The purity of the target protein was determined by 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of
the protein was measured by the Bradford method [17] using bovine serum albumin
as a standard.



Relative diadenylate cyclase activity assay
A total volume of 100 uL initial reaction mixture containing 100 mM CHES at
pH 9.5, 2 mM ATP, and 10 mM MgCl2 was used to detect the diadenylate cyclase

activity of btDisA. The reaction was initiated by adding 1 uM btDisA, and the reac-
tion mixture was incubated at 37 ? C for 30 min. The reaction was then terminated
by heating the reaction mixture in a boiling water bath for 10 min, and the mixture
was centrifuged at 16,000 × g for 10 min to remove the denatured protein. Sub-
sequently, the supernatant was loaded onto an Agilent 1200 series HPLC system
(Agilent Technologies, Santa Clara, CA), which contains a G1311A quaternary gra-
dient pump, a G1313A AutoSampler, and a G1314C UV detector  (wavelength was
set at 259 nm) to monitor the formation of c-di-AMP. The supernatant was sepa-
rated by an Elite Hypersil BDS C18 column (200 × 4.6 mm; 5 um particle sizes) with
a mobile phase of 90% phosphate buffer (30 mM K2HPO4 and 20 mM KH2PO4; pH
was adjusted to 6.0 using phosphoric acid ) and 10% methanol . The column temper-
ature was controlled at 25 ? C, and the flow rate was set at 1 mL/min. By injecting
10 uL of a series of 1–100 uM c-di-AMP standards into the HPLC, a regression equa-
tion, Y = 18921X + 2.6603 (R2 = 0.9997), of c-di-AMP was established in which Y and
X represent the peak area (mAU × s) and the concentration (uM) of the c-di-AMP
standard, respectively. The formation of c-di-AMP can be easily calculated using the
regression equation. The relative diadenylate cyclase activity was defined by divid-
ing the c-di-AMP amount produced at one condition by the maximum generated at
an optimized condition 



Identification of c-di-AMP by LC–ESI-MS/MS
A Finnigan Surveyor Plus liquid chromatography system followed by a Thermo
Scientific TSQ Quantum Ultra EMR tandem mass spectrum system (San Jose, CA, USA)
was used to confirm the product. Liquid chromatography separation was achieved
on a Thermo Scientific RP18 column (50 × 2.1 mm; 5 um particle size) [18]. The col-
umn was eluted at 35 ? C at a constant flow rate of 200 uL/min under the binary
mobile phase, which consisted of 10% methanol and 90% aqueous phase (containing
2 mM ammonium acetate and 0.1% formic acid). MS/MS parameters were as follows:
ionization source, electrospray ionization source; ionization mode, positive mode;
sheath gas pressure (N2), 30 units; auxiliary gas pressure (N2), 5 units; ion trans-
fer tube temperature, 270 ? C; collision gas pressure (Ar), 1.0 mTorr; spray voltage,
3000 V. The c-di-AMP was detected using the selected ion monitoring mode with
the two following m/z transitions: 659.00 ⇒ 312.54 at 32 eV and 659.00 ⇒ 330.31 at
18 eV.

Large-scale preparation and HPLC purification of c-di-AMP
For the enzymatic preparation of c-di-AMP, 2 uM btDisA was incubated with
100 mM CHES (pH 9.5) containing 10 mM ATP and 10 mM MgCl2 at 50 ? C in a 50 mL
reaction system for 4 h. The mixture was heated in a boiling water bath for 10 min
and then centrifuged at 16,000 × g for 10 min. The supernatant was concentrated
to approximately 5 mL and filtered through a 0.22 um filter. The concentrate was
loaded onto a Waters BondapakTM C18 semipreparative column (300 × 7.8 mm;

10 um particle size) in batches for the puri?cation of c-di-AMP on a Waters HPLC

system (Milford, MA, USA) consisting of a 515 pump, a Rheodyne 7725i injector with
a 200 uL injection loop, and a 2487 UV detector with the wavelength set at 259 nm.
The mobile phase consisted of 5% methanol and 95% aqueous phase (containing 0.2%
ammonium acetate; pH was adjusted to 6.0 using glacial acetic acid), and the flow
rate was 2 mL/min. The c-di-AMP containing fractions were pooled, concentrated
on a rotary evaporator, and finally lyophilized to yield a white powder.
For the determination of the purity of the produced c-di-AMP, 0.65 mg (defined
as the initial weight) of c-di-AMP sample was weighed and dissolved in 1 mL of
water. Before HPLC analysis, the c-di-AMP sample solution was diluted 20 times
to ensure that its concentration was in the concentration range of the c-di-AMP
standard series. Therefore, the purity of the c-di-AMP (P%) sample can be easily
calculated using the following equation:

 

where A, Mw, Dt, and Wi represent the peak area of the c-di-AMP sample, the molecu-
lar weight of c-di-AMP, the dilution factor, and the initial weight of c-di-AMP sample,
respectively.