Bacterial strains, chemicals and reagents
E.coli DH5α (Zymo Research, USA) was used as cloning host and BL21(DE3), C41(DE3) (Millipore Sigma (Novagen)) and CodonPlus(DE3) (Agilent Technologies) cells were used expression host strains. The pET28a expression vector (Thermo Scientific, USA), Taq DNA polymerase (G-Biosciences, USA), restriction enzymes (New England Bio Labs Ltd. UK), T4 DNA ligase (New England Bio Labs Ltd., UK) and molecular biology grade reagents were obtained from Sigma Aldrich (USA). His-Tag monoclonal antibody and secondary Anti mouse IgG (H+L) (Peroxidase/ HRP conjugated) were from Puregene: Genetix Biotech Asia Pvt. Ltd. respectively.
The non-directional cloning strategy was employed for the cloning of SigB (MSMEG_2752) gene of M. smegmatis mc2155. The primer pair used to amplify the SigB gene from the genomic DNA of M. smegmatis mc2155 were, forward primer SigB_2752_EcoRI 5’-CGGAATTCATGGCAAATGCCACCACAAGCC-3’and reverse primer SigB_2752_EcoRI 5’-CGGAATTCGGAGGCGTAGGAGCGGAGGCGG-3’respectively, where underline sequences are showing the EcoRI restriction site. The condition for PCR amplification were initial denaturation at 95°C for 5 minutes, 35 cycles (denaturation 95°C for 30 sec, annealing 60°C for 45 sec, elongation 72°C for 1 minute) and final elongation 72°C for 5 minute. The amplified PCR product was ligated into the EcoRI digested and dephosphorylated pET28a expression vector. The ligation product was transformed into DH5α cells and the recombinant positive clones (His-SigB) were confirmed by EcoRI restriction digestion and sequencing.
Expression of recombinant His-SigB protein
a. Expression host strain optimization
The positive clones containing His-SigB were transformed into three expression host strains of E.coli viz; BL21(DE3), C41(DE3) and CodonPlus(DE3) . These expression strains possess λDE3 lysogen that carries the gene T7 RNA polymerase under the control of lacUV5 promoter. So, IPTG (Isopropyl β-D-1-thiogalatopyranoside) induction for protein expression was performed to obtain maximum expression. The selected clone was inoculated from a single colony and cultured over night in 5 ml Luria Bertani (LB) containing 50 µg/ml kanamycin at 37°C in a shaking incubator. Next day, 1% (v/v) of secondary culture was incubated at 37°C until the OD600 of the culture reached ~0.4 O.D., the culture was induced with 0.1mM IPTG and grown for 3 hrs at 37°C at 150 rpm. For analysis of His-SigB protein expression, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by . For SDS-PAGE, 0.1 OD600 equivalent cell lysate prepared in 4X sample loading buffer (10% SDS, 40% glycerol, 0.25M Tris–Cl (pH 6.8), 200 mM DTT, and 0.05% bromophenol blue) after heat denaturation at 100°C were loaded onto the gel. For visualization, the gel was stained by Coomassie brilliant blue (R-250) followed by destaining.
b. IPTG optimization
Commonly, IPTG was used to achieve high level of expression for recombinant proteins. To optimize the concentration of IPTG on E. coli strains and the optimum protein expression, we selected best expressing host strain for a range of IPTG concentrations viz; 0.05, 0.1, 0.25, 0.5mM and induced at approximately < 0.4 OD600 at 37°C 150 rpm for 3 hrs. To determine the solubility of protein at various IPTG concentrations, the pellet/supernatant fractionation of the lysate was performed. The harvested cell cultures were washed and resuspended in 1X phosphate buffer saline (PBS, pH 8.0) and thereafter sonicated at 15% amplitude (ultrasonic homogenizer) for 2-3 min. The lysate was separated by centrifugation at 13,000 rpm for 20 min at 4°C containing soluble His-SigB in the supernatant as well as the pellet fraction resuspend in equal volume of the lysis buffer (1X PBS). The protein bands were visualized by denaturing SDS-PAGE after staining with Coomassie R-250 as mentioned above. Semi-quantitative analysis of the gels were performed with Quantity One software (BIO-RAD)
c. Temperature optimization
To optimize suitable temperature for protein expression, the best expressing host strain cultured at different temperatures viz; 37°C, 25°C, 16°C. The cells induced with 0.1mM IPTG at 37°C and 25 °C for 3 hours, while at 16°C for 12 hours with shaking at 150 rpm. The protein bands were visualized by SDS-PAGE and solubility fractionation analysis was done as above.
d. Time point optimization
To check best time point for the expression of His-SigB in the best expressing host strain, it was induced with 0.1 mM IPTG at 25°C/ 150 rpm for different time intervals viz; 0, 15, 30, 60, 120, 180 min. The expression profile was visualized in 10% SDS PAGE.
His-SigB purification and Immunoblotting
The recombinant His-SigB was purified by affinity using Ni-NTA beads as per manufacturer’s instructions. Briefly, the cells were lysed by sonication at 15% amplitude in Buffer A (Tris.Cl 50 mM (pH 8.0), NaCl 300 mM, Imidazole 10 mM, Triton X-100 1 % (v/v), PMSF 2 mM, Lysozyme 1 mg/ml) and the soluble lysate was clarified by centrifugation at 13,000 rpm (rcf 14,926 g) for 15 min. at 4°C. The pre-equilibrated Ni-NTA beads (Qiagen) in Buffer A was incubated with the lysate at 4°C for 30 mins., washed thrice with Buffer B (Tris.Cl 50 mM (pH 8.0), NaCl 300 mM, Imidazole 50 mM, Triton X-100 0.5 % (v/v)) and eluted thrice with Buffer C (Tris.Cl 50 mM (pH 8.0), NaCl 300mM, Imidazole 400 mM, Triton X-100 0.5 % (v/v)).
The purified His-SigB was resolved by SDS-PAGE and transferred to the PVDF membrane followed by blocking in 5% (w/v) skimmed milk powder (Himedia laboratories) in 1XPBST (Phosphate saline buffer containing 0.1% Tween-20). The blot was washed thrice by 1X PBST and incubated with anti-His antibody (0.5 µg/µl, 1:1000 in 1% PBST; Puregene) for 1 hr., washed with PBST and further incubated with secondary antibody anti-Mouse IgG (H+L Peroxidase/HRP conjugated, 1:5000 Pure gene Genetix Biotech) in PBST. Finally the blot was washed thrice by PBST, developed in ClarityTm ECL Bio-RAD, and analysed by ChemiDoc Imaging System (Bio-RAD).
Homology modeling and Structural comparison of SigA and SigB
The protein sequences of M. smegmatis sigma factors SigA and SigB were retrieved from National Center for Biotechnology Information (NCBI) database . To construct the homology models, the amino acid sequences of both the proteins were submitted to Phyre2 server . Phyre2 server utilizes multiple templates to produce the accurate model of the protein. In case of M. smegmatis SigA, three templates were used, namely, 5TW1(RNA polymerase sigma factor) , 6C05(crystal structure of RNA polymerase sigma factor from M. tuberculosis) , 4YG2 (X-ray crystal structure of sigma70 holoenzyme from E. coli)  with percent identities of 99%, 97% and 55% respectively. Similarly, M. smegmatis SigB model was build using two templates 5TW1 (RNA polymerase sigma factor) and 6C05 (crystal structure of RNA polymerase sigma factor from M. tuberculosis)  with percent identities of 65% and 64%, respectively. Both the models were validated on SAVES server using PROCHECK tool .
Protein-Protein docking of core RNA Polymerase (RNAP) and SigB
The protein-protein docking utilizes a multi-stage method for generating poses followed by their rankings. Here, we have used MOE platform  for the protein-protein docking experiment of core RNA Polymerase (RNAP) and SigB upon importing RNAP (PDB ID: 6EYD ) to MOE (https://www.chemcomp.com/Products.htm), workbench. Structure issues such as missing atoms, missing chains were automatically corrected using quick prep module. Optimal hydrogen positions and charges were optimized using protonated 3D module using the MOE software recommended settings. We have used here core enzyme of 6YED  PDB without sigma factor A (SigA). Before commencement of docking, bad crystallographic contacts or other imperfect geometries were overcome by the energy minimization method using Amber EHT 10 force field recommended by MOE platform. Energy Minimize application itself can be used to adjust hydrogens and lone pairs and to calculate partial charges. Similarly, SigB protein was prepared before docking. SigB model was considered as ligand and PDB ID: 6YED was considered as a receptor. After the docking run, the appropriate protein-protein fingerprints were generated for each pose which can be viewed using the PLIF visualization panel. Approximately, 100 coordinates were developed according to the binding energy. The best twenty dock complexes were considered for analysis.