Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis

Anbarasu Deenadayalan; Madhavan Dhanapal; Uma Devi Ranganathan; Ramalingam Bethunaickan

doi:10.3791/67680

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Method Article

Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis

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DOI:

10.3791/67680

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September 26th, 2025

Anbarasu Deenadayalan¹ , Madhavan Dhanapal¹ , Uma Devi Ranganathan¹^,² , Ramalingam Bethunaickan¹^,²

¹Department of Immunology, National Institute for Research in Tuberculosis, ²Faculty of Medical Research, Academy of Scientific and Innovative Research (AcSIR)

Summary

This protocol separates insoluble cell wall and membrane proteins into simple fractions (1-5 proteins) using preparative isoelectric focusing (IEF) based on isoelectric point, followed by separation by molecular weight. The resulting fractions can be used directly for immunological and proteomic analysis without further purification.

Abstract

Mycobacterium cell wall and membrane proteins, which play a central role in tuberculosis pathogenesis, were successfully separated using preparative Isoelectric Focusing (IEF) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Followed by gel elution, overcoming limitations in conventional methods for the separation of hydrophobic proteins. In this procedure, M. tuberculosis colonies were transferred from Lowenstein-Jensen slants into 2 mL of 7H9 broth, dispersed with glass beads, and incubated at 37 °C for 2 weeks. Then, the culture was scaled up to 200 mL and grown in a shaker for 4 weeks. It was further upscaled to 1 L with 500 mL of 7H9 broth and grown for an additional 4 weeks. Grown Mycobacteria were pelleted by centrifugation at 1741 × g for 30 min. For each 2 g pellet, 1 mL of breaking buffer was added, and the sample was sonicated. The lysate was centrifuged at 3436 × g for 15 min to remove unbroken cells, and the supernatant was concentrated. This supernatant (whole cell lysate) was centrifuged at 13751 × g for 30 min to pellet cell wall proteins. The remaining supernatant was ultra-centrifuged at 100,000 × g for 4 h to separate the cell membrane and cytosol. The isolated cell wall and membrane proteins were loaded onto a liquid preparative IEF system at 4 °C and separated at 12 W until the voltage stabilized at 1400 V, which separates 20 fractions. These IEF fractions were further separated by preparative SDS-PAGE, and proteins were eluted using a whole gel eluter at 250 mA, resulting in 30 fractions. Through this protocol, we were able to identify novel M. tuberculosis cell walls and membrane-specific biomarkers, and it also shows potential for characterizing similar proteins in other pathogens.

Introduction

Immunological studies have demonstrated that in addition to soluble proteins, which are easy to purify, the human immune response to pathogens such as M. tuberculosis is also directed hydrophobic protein present in the cell wall¹ and cell membrane proteins² of Mycobacterium tuberculosis. These cell wall and membrane proteins play a crucial role in immunological control against many pathogens, including tuberculosis. Some of these proteins are reported as immunodominant antigens that mount T and B cell response. In contrast, other proteins are identified as molecular signatures of pathogens that are reco....