Related Experiment Video
Updated: May 13, 2026
Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis
Published on: September 26, 2025
Differential detergent extraction of mycobacterium marinum cell envelope proteins identifies an extensively modified
Aniek D van der Woude1, Kozhinjampara R Mahendran, Roy Ummels
1Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Abstract:
A striking characteristic of mycobacteria is the presence of an unusual outer membrane which forms a thick permeability barrier and provides resistance to many antibiotics. Although specialized proteins must reside in this layer, only few mycolate outer membrane (MOM) proteins have been identified to date. Their discovery is complicated by difficulties in obtaining good separation of mycobacterial inner and outer membranes. During our efforts to identify novel mycobacterial outer membrane proteins (MOMPs), we discovered that we can enrich for MOMPs using differential solubilization of mycobacterial cell envelopes. Subsequently, these different fractions were analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This proteomic analysis confirmed that our marker proteins for inner membrane and MOM were found in their expected fractions and revealed a few interesting candidate MOMPs. A number of these putative MOMPs were further analyzed for their expression and localization in the cell envelope. One identified MOMP, MMAR_0617 of Mycobacterium marinum, was purified and demonstrated to form a large oligomeric complex. Importantly, this protein showed a clear single-channel conductance of 0.8 ± 0.1 ns upon reconstitution into artificial planar lipid bilayers. The most surprising feature of MMAR_0617 is a long C-terminal threonine-rich domain with extensive modifications. In summary, we have identified a novel mycobacterial outer membrane porin with unusual properties.
Insights
Researchers identified a novel mycobacterial outer membrane porin (MOMP) in Mycobacterium marinum. This protein, MMAR_0617, forms an oligomeric complex and exhibits unique channel properties, offering new insights into mycobacterial cell envelope structure.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Mycobacteria possess a unique outer membrane forming a permeability barrier and conferring antibiotic resistance.
- Identifying mycobacterial outer membrane proteins (MOMPs) is challenging due to difficulties in separating inner and outer membranes.
- Only a limited number of MOMPs have been characterized to date.
Purpose of the Study:
- To identify novel mycobacterial outer membrane proteins (MOMPs) using a novel enrichment strategy.
- To characterize the properties and localization of newly identified MOMPs.
- To investigate the functional and structural characteristics of a specific candidate MOMP.
Main Methods:
- Differential solubilization of mycobacterial cell envelopes to enrich for MOMPs.
- Proteomic analysis using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS).
- Expression and localization studies of candidate MOMPs.
- Purification and functional characterization of MMAR_0617 using artificial planar lipid bilayers.
Main Results:
- A novel enrichment method successfully isolated candidate MOMPs.
- Proteomic analysis identified several putative MOMPs, including MMAR_0617 from Mycobacterium marinum.
- Purified MMAR_0617 formed a large oligomeric complex and displayed single-channel conductance in lipid bilayers.
- MMAR_0617 possesses a unique, modified threonine-rich C-terminal domain.
Conclusions:
- A new strategy effectively enriches for mycobacterial outer membrane proteins.
- MMAR_0617 represents a novel mycobacterial outer membrane porin with unusual structural and functional characteristics.
- This discovery advances our understanding of the mycobacterial cell envelope and potential antibiotic targets.
