All chemicals were used in this study was analytical grade and used as received without any further purification. All solutions were prepared in milli-Q ultrapure water of resistivity not less than 18.2 MΩ cm-1.
Bacterial strains and plasmid
The bacterial strain Enteroinvasive Escherichia coli was gifted from NICED, Kolkata. All plasmid and expression vector used in this study were listed in Table no. 1A and 1B. All strains were handled in Biosafety level-II facility (Thermo Fisher Scientific A2 1300 Series).
Extraction of DNA and PCR amplification of IpaD gene
On Luria Bertani Broth Miller (Himedia, Mumbai, India), a single isolated colony of Enteroinvasive E.coli was cultured for 12 hours at 37°C with vigorous shaking at 200 rpm (Hazen et al. 2016). To collect the cell pellet, the culture was centrifuged at 13000 rpm for 5 minutes. The alkaline lysis procedure was used to isolate the plasmid DNA (Feliciello & Chinali 1993).Primers were generated using the IDT oligo analyzer tool and synthesized by IDT Technologies (India) for the amplification of the IpaD gene. To insert amplified IpaD gene into the pHis-TEV plasmid Vector, forward and reverse primers (Table No. 2) were constructed containing EcoRI and XhoI restriction endonuclease enzyme sites respectively. Gradient polymerase chain reaction was performed using the thermal Cycler (Biorad T100) to optimise the annealing temperature. A gradient temperature of 57.5°C to 61.5°C was set. The reaction was initiated by heating the reaction mixture at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s, and elongation at 68°C for 1 minute, and finally extension at 72°C for 10 minutes. Using the DNA agarose gel Electrophoresis Apparatus (Tarsons), the PCR product was analyzed in a 0.8% agarose (Sigma Aldrich) gel run at 70 V for 1 hour in 1X TAE (Tris-acetate-EDTA) buffer and bands were visualized using the UV Transilluminator (Himedia). The PCR product was purified using a Qiagen PCR Purification kit according to the manufacturer's instructions. The Nano-Drop Spectrophotometer (Eppendorf) was used to determine the DNA concentration.
Molecular cloning into expression plasmid and verification of the insert
The purified PCR product, i.e. IpaD gene and pHis-TEV plasmid vector were digested with EcoRI and XhoI restriction enzyme in digestion buffer (New England Biolabs) at 37°C for 2 hours as directed by the manufacturer and analysed on a 0.8% agarose gel. The T4 DNA ligase enzyme (New England Biolabs) was used to ligate the digested gene and plasmid vector, which was done at 16°C overnight as per the manufacturer's protocol. The resulted ligated product (pHis-TEV-IpaD) was transformed into Escherichia coli-DH5α competent cell following standard CaCl2 heat shock transformation protocol (Li et al. 2010) and the full construct of pHis-TEV containing IpaD gene was shown in (Fig. 1A). The positive colony was confirmed by double digestion analysis using EcoRI and XhoI restriction enzyme and colony PCR which were analyzed in a 0.8% agarose gel. The separated gene and vector fragmented part were visualized using the Gel Doc (Biorad) system and analyzed by Image Lab (Biorad) Software. The colony was maintained Luria Bertani agar (Himedia) plate containing ampicillin (100µg/ml) (Himedia) as selectable marker. For DNA sequencing, plasmid DNA was isolated from the positive colony using Qiagen Plasmid isolation kit as per manufacturer protocol. The sequencing was done by Integrated DNA Technologies (India) by the Sanger Sequencing method. NCBI Blast was used to analyse the sequence results.
IpaD protein expression optimization
Optimization of inducer concentration
Escherichia coli-BL21 (DE3) cells containing pHis-TEV-IpaD plasmid was grown overnight in Luria Bertani Broth supplemented with ampicillin (100 μg/ml). Five flasks containing fresh LB media were inoculated with overnight grown culture (1:100) and incubated at 37°C, 150 rpm, until the OD600 reached between ~0.7-0.8. The cultures were induced individually by five different isopropylthio-β-galactoside (IPTG) Concentrations, respectively 0.05mM, 0.25mM, 0.5mM, 1 mM and 2 mM for 18 hours at 15⁰C with 150 rpm. After induction, cells were harvested by centrifugation at 8000 rpm for 10 min at 4°C then the resultant pellet was resuspended in lysis buffer (20 mM Tris (Himedia) (pH 8.0), 500 mM NaCl (Himedia),10 mM imidazole (Himedia) and 5% sucrose (Himedia) in a 1:100 lysis buffer:culture volume ratio. Then cells were disrupted by sonication (5 cycles, 15-second pulse with 1-minute interval). To prevent protein degradation, 1mM Phenyl methyl sulfonyl fluoride (PMSF, Sigma Aldrich) and 1mg/ml lysozyme (Himedia) were added before sonication. The soluble and insoluble part were separated by centrifugation. The soluble fraction was collected in the supernatant portion by centrifuged at 20,000 rpm for 30 min at 4°C, and the insoluble part containing inclusion bodies was collected as pellet at bottom of the tube. The pellet was washed two times with a lysis buffer to remove any contaminant of soluble part. 8 M urea was added to the pellet to solubilize the insoluble inclusion bodies and boiled for 15 min, harvested by centrifugation. Protein expression and solubility were analysed by SDS -PAGE using ImageJ Software following two formulas.
Whereas, S is the amount of IpaD protein and I is the total protein after induced by IPTG; S’’ is the amount of the IpaD in supernatant fraction and P is the amount of total protein in pellet fraction
Optimization of post induction temperature
Escherichia coli-BL21 (DE3) cells harbouring pHis-TEV-IpaD plasmid were grown overnight with antibiotic containing LB media. From the overnight culture, three flasks containing fresh media were inoculated and kept at 37 ⁰C with 150 rpm until the OD600 reached ~0.7-0.8. After that, the effect of temperature on the expression of IpaD, culture was induced with different IPTG concentration and kept at three different temperatures such as 10⁰C, 15⁰C and 37⁰C for 18 hours. After the completion of incubation time, cells were harvested and disrupted by sonication, and expression was analyzed by densitometry analysis.
Purification of IpaD protein
Escherichia coli BL21 (DE3) containing pHis-TEV-IpaD plasmid was grown for 18 hours at 15° C with 150 rpm after induction with 0.5 mM IPTG. Then induced cells were collected by centrifugation and disrupted by sonication. Consequential supernatants were separated by centrifugation and filtered through a 0.45µm syringe filter unit (Himedia) to the trace amount of debris. Then, IpaD protein was purified by Ni-NTA affinity chromatography using the 5 ml His trap column (GE Health Care) as per manufacturer instructions with minor modification. Briefly, the column was equilibrated with 10 column volume (CV) equilibration buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM imidazole) containing 5% Sucrose. The supernatant was passed through the column four times for proper binding, and flow-through was collected. Afterwards, the column was washed twice with 10ml CV wash buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 5% Sucrose) with two different concentration of imidazole (20 mM imidazole, 60 mM imidazole) and wash fraction was also collected. The protein was eluted by applying the 5 ml elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 5% Sucrose) with three different concentration of imidazole (150 mM imidazole, 250 mM imidazole, and 500 mM imidazole). Then eluted fractions were immediately diluted with 1:1 ratio dilution buffer (20 mM Tris (pH 8.0), 500 mM NaCl) and were dialyzed to remove out the salt and imidazole against 1X TBS Buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5% sucrose) for 6 hours. The his tagged-IpaD protein was incubated with TEV protease overnight at 4°C (Sigma Aldrich) to remove the His-tag as described in the user manual provided by manufacturer. Further, the IpaD protein was applied to the His-trap column, and the flow-through fraction was collected as purified protein. Protein concentration was estimated using a Bradford reagent (Biorad) and analyzed by SDS -PAGE.
Analysis of protein stability
Purified IpaD protein (1mg/ml) was stored in cryotubes containing 1XTBS Buffer (20mM Tris, 150 mM NaCl pH 7.4) with 5% sucrose at 25°C (as room temperature), 4°C, -20°C and -80°C for 15 days. The secondary structure of IpaD protein was then analysed by Far UV CD.
SDS PAGE analysis and densitometry analysis
For SDS–PAGE analysis, 1X loading buffer (1M Tris-HCl (pH 6.8), 0.8 gm SDS, 10% glycerol, 14.7M β-mercaptoethanol,0.5M EDTA, 8mg bromophenol Blue) was added with the protein samples and was heated for 5 min at 95⁰ C. Protein samples were run in 15% resolving and 4% stacking gels for 2 hours at 120 V (Biorad mini protein system). The gel was stained with Coomassie blue (50% methanol, 10% acetic acid and 0.1% Coomassie brilliant blue R-250) thereafter destanied with (45% methanol and 10% acetic acid). A prestained ladder (Biorad) was used as standard. For densitometry analysis, the area under the curve (AUC) of the protein was analyzed from the recorded image by using molecular imager (Biorad), and images were analyzed by ImageJ software.
Western blot analysis
For western blotting after resolving the IpaD protein sample was transferred onto the PVDF membrane (Millipore) using semidry transfer apparatus (Biorad) for 1 hour with constant voltage at 20V. After complete transfer membrane was washed with TBST buffer (20 mM Tris pH7.5, 150mM NaCl and 0.1% (v/v) Tween-20 (Sigma) and then blocked with blocking buffer (3% BSA in TBST buffer) for 1 hour at room temperature with gentle shaking. The membrane was then incubated with mouse anti-IpaD monoclonal antibody (1:1000, Abbexa, USA) in TBST containing 1% BSA overnight at 4 °C. Next, the membrane was washed for three times with TBST then incubated with anti-mouse secondary antibody conjugated with HRP (IgG-HRP) (1:5000; Invitrogen, USA) for 1 hour at room temperature in 3% (w/v) BSA in TBST. After washing thrice with TBST, immunoblot was developed in the presence of a chemiluminescence substrate (Bio-Rad).
Size exclusion chromatography
IpaD was further concentrated using Amicon ultrafilter (Millipore) and purified by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare). Column was equilibrated using the mobile phase as TBS Buffer (20mM Tris, 150mM NaCl pH-7.4) containing 5% sucrose. The flow rate was maintained at 0.4ml/min, and UV detection was set at 280 nm. 500 µg protein was loaded with injection volume of 500µl. Eluted purified was analysed by SDS PAGE.
Circular dichroism analysis
For secondary structure analysis, Far-UV CD was performed using J-815 Spectrophotometer (Jasco). Three Spectra was measured between the 250-200 region with scan speed 50nm/min and bandwidth 1 nm. 1X TBS (20mM Tris, 150mM NaCl pH-7.4) buffer containing 5% sucrose was used as control for the analysis.
Statistical analysis was performed by using Student’s t test to calculate significance of differences. All experiments were accomplished at least three replicates and the data are presented as the mean ± standard deviation (SD).The values of *p < 0.05 represents significant, **p < 0.01and ***p < 0.001 represents very significant.