ClpC operon regulates cell architecture and sporulation in Bacillus
anthracis
Lalit K. Singh*, Neha Dhasmana*, Andaleeb Sajid, Prasun Kumar, Asani Bhaduri,
Mitasha Bharadwaj, Sheetal Gandotra, Vipin C. Kalia, Taposh K Das¶, Ajay K.
Goel§, Andrei P. Pomerantsev†, Richa Misra , Ulf Gerth±, Stephen H. Leppla† and
Yogendra Singh
Figure S1 A.
Figure S1 A. ClpC operon organization in B. anthracis. ClpC operon comprising
of four genes: ctsR, mcsA, mcsB and clpC. Deletions were made by homologous
recombination using intragenic regions of respective genes. All deletions were inframe maintaining normal expression of downstream genes in the operon.
Figure S1 B
Figure S1 B. Confirmation of mutants by polymerase chain reaction. Genomic
DNA was isolated from Bacillus anthracis Sterne and mutant strains. The gene
encoding for CtsR, McsA, McsB, ClpC and whole operon were amplified using
gene specific primers (Table S1). The size of the amplified product was reduced in
mutant strain and corresponded to expected size of the genes.
Figure S1 C
KDa
175
90
60
40
30
20
10
ClpC protein
Figure S1 C. Confirmation of in-frame deletion in mutant strains of Bacillus
anthracis. Expression of ClpC (upper panel) and western blot developed using antiClpC antibodies (lower panel) using whole cell lysate of each mutant. GST is a
glutathione S-transferase protein used as negative control.
Figure S2
Verification of complemented strains of Bacillus anthracis by PCR. PCR was
performed using specific primers for mcsB and clpC. Lane-1: DNA ladder (1-Kb); lane-2:
mcsB gene amplification from genomic DNA of wild type B. anthracis Sterne; lane-3:
mcsB gene amplification from genomic DNA of ΔmcsB strain; lane-4: mcsB gene
amplification from mcsB complemented strain; lane-5: clpC gene amplification from
genomic DNA of wild type B. anthracis Sterne; lane-6: clpC gene amplification from
genomic DNA of ΔclpC strain; lane-7: clpC gene amplification from clpC complemented
strain; and lane-8: DNA ladder (100-bp)
Figure S3
A
B
Figure S3. continued…
C
D
Phase contrast microscopy of wild type Bacillus anthracis Sterne and mutant strains.
Cells were grown up to exponential phase in LB broth, diluted to OD595 = 0.035, plated on
sporulation medium (in 3% low melting point agarose) and incubated at 30 °C. Time lapse
imaging (1000 × magnification with oil immersion) was performed on phase contrast
microscope (Nikon Ti Eclipse). Wild type (A); ΔmcsB (B); ΔclpC (C) and Δoperon (D).
Figure S4.
A
B
Scanning electron microscopy of vegetative cells of Bacillus anthracis. Cells were
grown at 37 °C in LB broth and harvested at mid-log phase. Cells were washed with 0.1 M
sodium phosphate buffer and fixed primarily with Karnovasky’s fixative and then treated
with 1% osmium tetroxide. The samples were dehydrated, subjected to critical point drying
and coated with gold. Cells were visualized under Zeiss EVO LS15. ΔclpC (A) and
Δoperon (B).
Table S1. List of primers used in this study
Primer Name
Primer sequence 5´→ 3´
Primers for knockout generation
L CtsR Fp
CCCTCAGAATGGTTGGAATTCATTCGTAGAGTGC (EcoR I)
L CtsR Rp
GATATTGCTCAATGATACTAGTTATATTTCTCATTTATCC (Spe I)
R CtsR Fp
CTTAGGGCTCGAATTCTATGCGCAATGTTAAG (EcoR I)
R CtsR Rp
GCTGATTTTCAAGATTACTAGTTTTATCCCTTACTTC (Spe I)
L McsA Fp
CTAAAGCAAGTTATTGAATTCAGTAATAATAATGTG (EcoR I)
L McsA Rp
CTTCTTCTCGTTGACTAGTTTTGTATAATG (Spe I)
R McsA Fp
GAACTAAAACTAGTTCTGAAACAATACG (Spe I)
R McsA Rp
GTTGAATAACACTAGTTAAATCTGCAATAATATC (Spe I)
L McsB Fp
GGGGATAGTGATGAATTCTCAAAACTGTAATATAAG (EcoR I)
L McsB Rp
CTTCATCCATGGACTAGTCGCTTCATTC (Spe I)
R McsB Fp
CACAACCAGGAATTCTACAACAATATG (EcoR I)
R McsB Rp
GATGGTTTTAAACTAGTCGATGCGTCAATAG (Spe I)
L ClpC Fp
GTTTTAAGTAGTCGAATTCGTTTGGC (EcoR I)
L ClpC Rp
CTTGAGATAAAGCTACTAGTTTCTGTGCTCTTTCTG (Spe I)
R ClpC Fp
GCCTTCCGTCCAGAATTCTTAAACCG (EcoR I)
R ClpC Rp
GATTACTACCATTTACTAGTAGACGATCGGCACGC (Spe I)
L Operon Fp
CCCTCAGAATGGTTGGAATTCATTCGTAGAGTGC (EcoR I)
L Operon Rp
GATATTGCTCAATGATACTAGTTATATTTCTCATTTATCC (Spe I)
R Operon Fp
GCCTTCCGTCCAGAATTCTTAAACCG (EcoR I)
R Operon Rp
GATTACTACCATTTACTAGTAGACGATCGGCACGC (Spe I)
Primers for knockout confirmation
CtsR Fp
ATGAGAAATATATCTGATATCATTGAGCAAT
CtsR Rp
TTATTTATATTTCAGTGTTCTTAACATTGCGC
McsA Fp
ATGACTTGTCAAAACTGTAATATAAGACCAGC
McsA Rp
CTATTCCCCCTCTCTATACTCACTAAGC
McsB Fp
ATGTCACTGGACAAAATTATGAATGAAGCG
McsB Rp
TTAGTTTTTTTCAATACGTAATCGCTCACG
ClpC Fp
ATGATGTTTGGAAGATTTACAGAAAGAGCACAGAAAG
ClpC Rp
TTATTTTACCTTTTCGGCACTATGAATGACAAATG
Primers for gene cloning
pProExHTc-McsB Fp
GAGAGGGGGAGGATCCCTATGTCACTGGACAAAATTATGAATG (Bam HI)
pProExHTc-McsB Rp
AGAAATCGCCTCAAGCTTGCGCTTAGTTTTTTTCAATACGTAATCGC (Hind III)
pProExHTc-ClpC Fp
GCAAGTAGGAGGGGATCCCTATGATGTTTGGAAGATTTACAG (Bam HI)
pProExHTc-ClpC Rp
GCCCTCTTAGTTTCTCGAGACTTATTTTACCTTTTCGGCAC (Xho I)
Primers for complementation
pYS5-SDM Fp
GGCATTGTCTACGATACTAGTTTCAAGCACAGG (Spe I)
pYS5-SDM Rp
CCTGTGCTTGAAACTAGTATCGTAGACAATGCC (Spe I)
pYS5-SDM Fp
GGGGATTTTATGCGTGAGAATGGTACCGTCTATCCCG (Kpn I)
pYS5-SDM Rp
GGCAATGCCGGGATAGACGGTACCATTCTCACGCATA (Kpn I)
ClpC Promoter Fp
CCGAACACGGTACCTAAGCTCTCTAGCG (Kpn I)
ClpC Promoter Rp
ATCAGATACTAGTCTCATTTATC (Spe I)
pYS5-McsB Fp
GAATAGTTCTATGTCACTAGT CAAAATTATGAATG (Spe I)
pYS5-McsB Rp
CATCATAG AAATCGGATCCTACTTGCGCTTAG (Bam HI)
pYS5-ClpC Fp
GATTTCTATGATGTTTGG ACTAGTTACAGAAAGAGCA (Spe I)
pYS5-ClpC Rp
GCCCTCTTAGTTTGTGGATCCTTATTTTACC (Bam HI)
Table S2. List of plasmids used in this study
Name
Description
Resistance Marker
Reference
pSC
Plasmid used for single crossovers in B. anthracis; AmpR
in E. coli; EmR both in E. coli and B. anthracis
Erythromycin, Ampicillin
Andrei P.
Pomerantsev, 2009
pSC-L-ctsR
Left fragment of ctsR was cloned for first crossover
Erythromycin, Ampicillin
This study
pSC-R-ctsR
Right fragment of ctsR was cloned for second crossover
Erythromycin, Ampicillin
This study
pSC-L-mcsA
Left fragment of mcsA was cloned for first crossover
Erythromycin, Ampicillin
This study
pSC-R-mcsA
Right fragment of mcsA was cloned for second crossover
Erythromycin, Ampicillin
This study
pSC-L-mcsB
Left fragment of mcsB was cloned for first crossover
Erythromycin, Ampicillin
This study
pSC-R-mcsB
Right fragment of mcsB was cloned for second crossover
Erythromycin, Ampicillin
This study
pSC-L-clpC
Left fragment of clpC was cloned for first crossover
Erythromycin, Ampicillin
This study
pSC-R-clpC
Right fragment of clpC was cloned for second crossover
Erythromycin, Ampicillin
This study
pSC-L-operon
Left fragment of operon was cloned for first crossover
Erythromycin, Ampicillin
This study
pSC-Roperon
Right fragment of operon was cloned for second
crossover
Erythromycin, Ampicillin
This study
pCrePAS-71
Contains cre gene and strongly temperature-sensitive
replicon for both E. coli and gram-positive bacteria; SpR
in both E. coli and B. anthracis.
Spectinomycin
Andrei P.
Pomerantsev, 2006
pYS5
Plasmid used for complementation in B. anthracis;
Kanamycin, Ampicillin
Yogendra Singh, 1989
AmpR in E. coli; KanR in B. anthracis
pYS5-mcsB
Complement plasmid expressing McsB under native
promoter
Kanamycin, Ampicillin
This study
pYS5-clpC
Complement plasmid expressing ClpC under native
promoter
Kanamycin, Ampicillin
This study
pProExHTc
E. coli expression vector with N-terminal His6-tag
Ampicillin
Invitrogen
pProExHTcclpC
Expression of His6-ClpC in E. coli
Ampicillin
This study
pProExHTcmcsB
Expression of His6-McsB in E. coli
Ampicillin
This study
Table S3. Strains used in this study
Name
Resistance
marker
References
E. coli B strain: F-, dcm, ompT, hsdS(rB- mB-),
gal,λDE3(lacI lacUV5-T7 gene 1, ind1, sam7,
nin5)
-
Invitrogen
DH5α
E. coli fhuA2, Δ(argF-lacZ)U169, phoA glnV44,
Φ80, Δ(lacZ)M15, gyrA96, recA1, relA1, endA1,
thi-1, hsdR17
-
Invitrogen
SCS110
E.coli SCS110 is an endA– derivative of the
JM110 strain rpsL (Strr) thr leu endA thi-1 lacY
galK galT ara tonA tsx dam dcm supE44 Δ(lacproAB) [F´ traD36 proAB lacIqZΔM15]
-
Stratagene
B. anthracis strain pXO1+, pXO2-
-
NIAID, NIH
𝛥ctsR
B. anthracis Sterne:: ctsR-
-
This study
𝛥mcsA
B. anthracis Sterne:: mcsA-
-
This study
𝛥mcsB
B. anthracis Sterne:: mcsB-
-
This study
𝛥clpC
B. anthracis Sterne:: clpC-
-
This study
B. anthracis Sterne:: clpC operon-
-
This study
BL21(DE3)
B. anthracis Sterne 34F2
𝛥operon
Genotype
mcsB complement
B. anthracis Sterne:: mcsB- + pYS5-mcsB
Kanamycin
This study
clpC complement
B. anthracis Sterne:: clpC- + pYS5-clpC
Kanamycin
This study
Detailed Materials and Methods
Generation of B. anthracis Sterne clpC Operon Gene Mutants and their
Characterization.
To understand the role of clpC operon in the physiology of B. anthracis Sterne strain, the
cre-lox genetic modification method was used to introduce precise genetic knockouts as
reported earlier by Pomerantsev et al., (2006). In brief, the pSC vector was used to produce
a deletion in each gene or whole operon (Figure S1 A) by two step recombination using the
primers shown in Table S1. Plasmid pCrePAS was used for the elimination of DNA regions
containing a spectinomycin resistance cassette located between two similarly oriented loxP
sites. The deleted region of gene in each mutant strain was confirmed by DNA sequencing
and PCR (Figure S1 B). The expression of ClpC protein in each mutant showed that all
mutants had in-frame deletions (Figure S1 C).
Whole Cell Lysate Preparation and Western Blot Analysis
All mutant strains were grown at 37 °C for overnight and harvested at 12,000 × g for 10
min. Cells were suspended in sonication buffer [50 mM Tris-HCl (pH8.5), 5 mM βmercaptoethanol, 1 mM phenylmethylsulfonylfluoride (PMSF), 1× protease inhibitor
cocktail (Roche Applied Science, USA) and 300 mM NaCl] and sonicated for 5 min. The
lysed cells were centrifuged at 12,000 × g for 30 min. The supernatant was aliquoted and
stored at 80 °C. Total protein in cell lysates were estimated using Bradford reagent and 25
μg of each lysate was loaded on 12% SDS-PAGE and transferred on nitrocellulose
membrane (Millipore, USA) followed by blocking with 3% bovine serum albumin
(SigmaAldrich, USA). Primary antibody against ClpC protein was raised in rabbit and
used at 1:30,000 titer. HRP-conjugated secondary antibody against rabbit was used at
1:20,000 dilution. Blots were developed using Super-signal West Pico Chemi-Luminescent
substrate (ThermoFisher Pierce, USA). Controls used were purified ClpC protein and
GST protein.
Complement Strain Preparation.
Plasmid pYS5, a shuttle vector of E. coli and B. anthracis, has protective antigen gene
under its native promoter (3). We created SpeI and KpnI restriction enzyme sites using
Quick-change site-directed mutagenesis kit as per manufacturer’s instruction (Stratagene,
USA) and primers (Table S1). SpeI restriction enzyme site was created within the signal
sequence 39-bp downstream from the +1 ATG starting codon of protective antigen signal
sequence while the KpnI restriction enzyme site created 297-bp upstream to the +1 ATG
codon of PA signal sequence spanning the PA promoter region. Both the genes (mcsB and
clpC) were amplified from the wild type genomic DNA with mcsB forward primer (SpeI),
clpC forward primer (SpeI) and mcsB reverse primer (BamHI) and clpC reverse primer
(BamHI). The promoter region of clpC operon were amplified from the wild type genomic
DNA with forward primer (KpnI) and reverse primer (SpeI). The genes were cloned in the
pYS5 (SpeI+BamHI digested) followed by the cloning of clpC operon promoter
(KpnI+SpeI digested). The electrocompetent cells were prepared as described by Quinn and
Dancer (1990). The plasmid containing mcsB and clpC genes were electroporated using
BTX Electro cell Manipulator 600 (1KV, 186 ohms, 25µF using 0.1 cm Bio-Rad Electrocuvette) in SCS110 strain. The plasmids were isolated from SCS110 using Qiagen
Miniprep kit (USA) and electroporated in respective knockouts electro-competent cells.
The complementation was confirmed by PCR (Figure S2).
Bacillus anthracis Spore Preparation.
The wild type and knockout strains were grown in 10 mL Luria-Bertani (BD Difco, USA)
broth up to mid-log phase (OD595 = 0.8). Each culture was inoculated into 100 mL
sporulation medium containing 0.6 mM CaCl2.2H2O, 0.8 mM MgSO4.7H2O, 0.3 mM
MnSO4.H2O, 85.5 mM NaCl, 0.025 mM ZnSO4, 0.02 mM CuSO4, and 8 g of LB broth/L
(pH6.0) as described earlier (Barua et al., 2009). Cultures were incubated at 30 °C for 72
h on constant shaking at 200 rpm. The spores were harvested at 12,000 × g for 15 min at 4
°C and transferred to 50 mL autoclaved milliQ water to complete sporulation at 30 °C at
200 rpm. Spores were harvested at 120 h and washed thrice with autoclaved milliQ water
and finally re-suspended in 5 mL milliQ water. Spores were heat activated at 65 °C for 30
min and stored at 20°C. Sporulation and germination efficiencies were calculated as
described by Giebel et al., (2009) and Burns et al., (2010).
Phase Contrast Time Lapse Imaging
Bacillus mutant strains were grown in chemically defined media [62 mM K2HPO4, 44 mM
KH2PO4, 15 mM (NH4)2SO4, 6.5 mM Sodium Citrate, 6.5 mM MgSO4, 2.2 mM Glucose, 6
μM L-glutamic acid, 7.5 μM MnCl2, 0.15 × Metal Mix, pH7.4] and diluted OD595 = 0.035.
Sporulation medium supplemented with 1.5% high-resolution low-melting agarose pad was
prepared on Corning Micro Slide Frosted (75 × 25 mm) with the help of AB gene-frame
(17 × 54 mm). One μl of culture was spread on agarose pad sealed with ESCO cover slips
No.1 thickness (24 × 60 mm). The cells were incubated at 30 °C for 24 h to 144 h in moist
condition. Imaging was done at different time intervals on Nikon ECLIPSE TE 2000-S with
phase III at 1000× magnification in oil immersion (Figure S3) (De Jong et al., 2011).
Transmission Electron Microscopy.
B. anthracis Sterne and deletion mutants were grown at 37 °C in LB Broth and harvested at
mid log phase. The cells were pelleted at 12,000 × g for 10 min and washed three times
with 100 mM sodium phosphate buffer (pH7.4). The samples were immediately
transferred for primary fixation to Karnovasky’s fixative [2.5% Glutaraldehyde (TAAB,
UK) + 2% Paraformaldehyde (SigmaAldrich, USA) in 100 mM sodium phosphate buffer
pH7.4] for 12 h at 4 °C. The samples were rinsed thoroughly with sodium phosphate
buffer to wash off the un-reacted excess fixative. Secondary fixation was performed with
1% osmium tetroxide (TAAB, UK) for 1 h at 4 °C and then dehydrated with graded acetone
(Merck Chemicals, Germany) [30%, 50%, 70%, 80% (×2), 90%, 100% (×3); 30 min each
at 4 °C]. Absolute xylene (Merck Chemicals, Germany) was used for clearing process and
dehydrating agent was replaced from the samples. The bacterial samples were in-filtered
and embedded into araldite resin mixture (TAAB, UK). Infiltration was carried out by
gradually decreasing the concentration of clearing agent and increasing the concentration of
embedding medium proportionately. Curing was processed at 55 °C for 24 h and then 65
°C for 48 h. Sectioning was performed with Leica UC6 ultra-cut and grids were observed in
FEI Tecnai G2 Spirit at 200 KV (Putnam et al., 2013).
Scanning Electron Microscopy.
The deletion mutants and wild type strains were grown at 37 °C in LB broth and harvested
at mid-log phase and processed as earlier described by Dubey et al., (2011). The cells were
pelleted at 12,000 × g at 4 °C, washed three times with 100 mM sodium phosphate buffer
(pH7.4). The samples were fixed with Karnovasky’s fixative (2.5% Glutaraldehyde + 2%
Paraformaldehyde in 100 mM sodium phosphate buffer pH7.4) for 6 h at 4 °C and again
washed with sodium phosphate buffer thrice. Secondary fixation was performed with 1%
osmium tetroxide for 10 min at 4 °C and then dehydrated with graded ethanol [30%, 50%,
70%, 80% (×2), 90%, 100% (×3); 30 min each at 4 °C]. Samples were dried with a critical
point drying technique and subjected to the gold coating of 10 nm thickness with an Agar
sputter coater on alumimium stubs. Samples were observed in Zeiss Scanning Electron
Microscope EVO LS15 at 20 KV. Comprehensive imaging, processing and analysis
performed on Smart SEM software.
Ni-NTA Protein Purification and Antibody Generation.
The genes encoding for clpC were PCR amplified using B. anthracis Sterne genomic DNA,
using primer pair pProEx-HTc-McsB Fp and pProEx-HTc-McsB Rp, pProEx-HTc-ClpC Fp
and pProEx-HTc-ClpC Rp (Table S1). The amplicons thus generated were digested with
BamHI and XhoI restriction enzymes respectively and ligated into pProEx-HTc previously
digested with similar enzymes. The integrity of both plasmid constructs was confirmed by
DNA sequencing. The recombinant His6-tagged proteins were purified by metal affinity
protein purification. Escherichia coli BL21 (DE3) cells were transformed with pProExHTc-McsB and pProEx-HTc-ClpC plasmids expressing full-length proteins.
The
recombinant E. coli strains harboring the pProEx-HTc-McsB/ClpC plasmid was used to
inoculate 1L of LB broth supplemented with ampicillin 100 𝜇g/mL followed by incubation
at 37 °C with shaking (220 rpm) until A595 reached 0.8. The culture was induced with IPTG
at concentration 1 mM and growth was continued for additional 1216 h at 16 °C. Cells
were harvested at 10,800 × g for 10 min and stored at 80 °C.
Pellets were thawed on ice and suspended in cell lysis buffer [50 mM Tris-Cl (pH8.5),
300 mM NaCl, 5 mM β-mercaptoethanol, 1 mM PMSF and 1× protease inhibitor cocktail
(Roche Applied Science, USA)] and lysed by sonication. The cell lysates were centrifuged
at 30,000 × g for 30 min. The supernatant containing recombinant protein was collected and
incubated for 2 h with Ni2+-nitrilotriacetic acid resin (Qiagen, USA) pre-equilibrated with
buffer [50 mM Tris-Cl (pH8.5), 300 mM NaCl, 10% glycerol, 20 mM imidazole, 10 mM
β-mercaptoethanol and 1 mM PMSF]. After extensive washings with buffer A, resin was
further washed with high salt containing buffer [50 mM Tris-Cl (pH8.5), 1 M NaCl, 10%
glycerol, 20 mM imidazole, 10 mM β-mercaptoethanol, and 1 mM PMSF]. Elution was
carried out in elution buffer [50 mM Tris-Cl (pH8.5), 150 mM NaCl, 10% glycerol, and
200 mM imidazole]. Protein fractions were run on 10% SDS-PAGE and stained with
coomassie brilliant blue (Gupta et al., 2009). Purified fractions were pooled, dialyzed [20
mM Tris-Cl (pH8.0) 10% glycerol and 150 mM NaCl], aliquoted and stored at 80 °C.
Rabbits were immunized with 500 g antigen in Freund’s complete adjuvant
(SigmaAldrich, USA) at day one. Subsequently five booster doses of 200 g antigen in
Freund’s incomplete adjuvant were given at 15th, 25th, 35th, 45th and 55th day. Serum was
collected on 75th day and titer was determined by direct ELISA.
Polyhydroxybutyrate Assay during Sporulation.
Primary inoculum for PHB production was prepared using single bacterial colony from LB
agar plate into 10 mL LB broth, at 37 °C for 1620 h at 200 rpm. It was used as inoculum
in sporulation
medium (0.6 mM CaCl2.2H2O, 0.8 mM MgSO4.7H2O, 0.3 mM
MnSO4.H2O, 85.5 mM NaCl, 0.025 mM ZnSO4, 0.02 mM CuSO4 and 8 g of LB broth/L,
pH6.0). PHB production was monitored in mcsB, clpC and operon deleted mutants and
compared to B. anthracis Sterne as control. Cultures were grown in 200 mL sporulation
medium by incubating at 37 °C and 200 rpm. Aliquots of 100 mL were analyzed for cell
dry weight (CDW) and PHB content at various time periods upto 144 h after inoculation.
Samples (100 mL) were centrifuged at 8,000 × g at 4 °C for 20 min. The pellet was washed
with 10 mL saline solution (0.9 % NaCl). The pellet was dried at 85 °C for 36 h and
weighed to estimate CDW. Cell pellet (40 mg) was processed and treated for propanolysis.
Propyl-esters of β-hydroxyacids from PHB hydrolysis were analyzed by gas chromatograph
(GC) equipped with dimethylpolysiloxane capillary column DB-1 (30 m × 0.25 mm × 0.25
µm) and flame ionization detector by the method described earlier (Patel et al., 2012).
Assay of Anthrax Lethal Toxin in Supernatants.
Wild type and deletion mutants were inoculated in 10 mL LB broth and incubated
overnight at 37 °C. Secondary inoculation (10%) was performed in 50 mL Brain-Heart
Infusion media (BD Difco, USA) containing 10% sodium bicarbonate. The cultures were
incubated at 37 °C for 4 h at 200 rpm shaking. The cultures were pelleted at 12,000 × g for
30 min and supernatants were collected followed by their filter sterilization. Precipitation
was performed by adding 20% trichloroacetic acid (Merck Chemicals, Germany) and
incubating at 20°C for 1 h. Proteins were pelleted at 12,000 × g for 30 min at 4 °C. Protein
pellets were washed thrice with 5 mL ice-chilled acetone. The pellets were dried at 95 °C
and re-suspended in 1 mL PBS containing 1× protease-inhibitor cocktail (Roche Applied
Sciences, USA). The resuspended protein pellets were loaded along with purified protective
antigen/lethal factor on 12% SDS-PAGE followed by western blotting.
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