In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria

Krishna K Singh; Devendra P Singh; Rambir Singh; Deepak K Saini

doi:10.21769/BioProtoc.3291

In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria

Bio Protoc. 2019 Jul 5;9(13):e3291. doi: 10.21769/BioProtoc.3291.

Authors

Krishna K Singh^{1

2}, Devendra P Singh¹, Rambir Singh², Deepak K Saini^{1

3}

Affiliations

¹ Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
² Institute of Biomedical Sciences, Bundelkhand University, Jhansi, India.
³ Centre for Biosystems Science and Engineering, Indian Institute of Science, Bangalore, India.

Abstract

Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic intermediates. In this protocol, using response regulator proteins as targets we describe the experimental strategy for probing the occurrence of acetylation using purified recombinant proteins in an in vitro setup. Further using M. smegmatis strains overexpressing the wild type or mutant response regulator protein, we also describe how in vivo acetylation can be validated in Mycobacterial proteins. The described approach can be used for analyzing acetylation of any mycobacterial protein under both in vitro and in vivo conditions.

Keywords: Acetylation; KATms; M. tuberculosis; Post-translational modification; Response regulator; TcrX; Two-component signaling.