Research Article Expression, Purification, and In Silico Characterization of Mycobacterium smegmatis Alternative Sigma Factor SigB Rakesh Kumar Singh, 1 Lav Kumar Jaiswal, 1 Tanmayee Nayak, 1 Ravindra Singh Rawat, 2 Sanjit Kumar, 2 Sachchida Nand Rai , 3 and Ankush Gupta 1 1 Molecular Microbiology Laboratory, Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi- 221005, Uttar Pradesh, India 2 Centre for Bioseparation Technology, Vellore Instiute of Technology, Vellore-632014, Tamil Nadu, India 3 Centre for Biotechnology, University of Allahabad, Prayagraj-211002, Uttar Pradesh, India Correspondence should be addressed to Ankush Gupta; ankushgupta@bhu.ac.in Received 28 February 2022; Accepted 5 May 2022; Published 20 May 2022 Academic Editor: Pei-Wen Zhu Copyright © 2022 Rakesh Kumar Singh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA (σ 70 ) but dispensable for growth in both Mycobacterium smegmatis (Msmeg) and Mycobacterium tuberculosis (Mtb). It is involved in general stress responses including heat, oxidative, surface, starvation stress, and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive extra cytoplasmic function (ECF) sigma factors (SigH, SigE, SigL) and SigF involved in multiple signaling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of E. coli, viz., (BL21 (DE3), C41 (DE3), and CodonPlus (DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time duration. We achieved the maximum expression of SigB at 25 ° C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA that is carried out using homology modeling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Msmeg and SigB. This effort provides a platform for pulldown assay, structural, and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signaling networks along with its regulation. 1. Introduction Prokaryotic transcription is mediated by RNA polymerase (RNAP) which is made up of five core subunits (α 2 ββ ′ ω) that binds with the sigma factor (σ), also known as tran- scription initiation factor that provides specificity during transcription initiation. Based on the structure and function, σ factors are classified into two major groups, viz., (i) sigma 70 (σ 70 ), i.e., primary sigma and (ii) sigma 54 (σ 54 ) which is involved in nitrogen fixation and found mostly in plant growth promoting rhizobacteria. Further, σ 70 is classified into four major groups based on the domain organization, viz., groups 1, 2, 3, and 4. Group 1 (SigA) has an extended N-terminal region and four highly conserved regions, namely, regions 1, 2, 3, and 4. The group 2 (SigB) does not have the N-terminus extended region, but all the four con- served regions are present. The group 3 (SigF) has only three regions, viz., 2, 3, and 4 while the group 4 (also known as the Extra Cytoplasmic Function (ECF) sigma factor) contains only regions 2 and 4. The regions 2 and 4 are extremely important in promoter recognition and binds at -10 and -35 regions of the promoter, respectively [1, 2]. σ 70 being the principle σ factor contributes in the expres- sion of all housekeeping genes under normal conditions [3, 4]. However, under various stressful conditions, alternative sigma factors (ASF) redirect the transcription machinery Hindawi Disease Markers Volume 2022, Article ID 7475704, 11 pages https://doi.org/10.1155/2022/7475704