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

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.

Our research focused on developing a streamlined method to separate insoluble cell wall and cell membrane proteins of Mycobacterium tuberculosis into simple fractions for direct proteomic and immunological assays. Advances in the electrophoretic techniques now allow complex proteins to be separated into fewer protein fractions, facilitating efficient mass spec-based isolation for functional proteomic and immunological applications. Key challenges include poor solubility of hydrophobic protein precipitating above hundred milligram and SDS-PAGE induced denaturation, which hinders downstream application needing native confirmation like enzyme activity assays.

No current protocol systematically separates insoluble hydrophobic cell wall and cell membrane proteins into defined water-soluble fractions for direct proteomic and immunological analysis, which is key to understanding function and guiding interventions. Our future research will focus on developing T-cell antigen-based diagnostics and to evaluate their sensitivity, specificity, and performance across the TB population for an early, accurate TB detection with an aim to support better disease control. To begin, obtain the cell wall, cell membrane, and cytosolic protein samples from whole cell lysate of Mycobacterium tuberculosis bacilli.

Solubilize the cell wall and cell membrane proteins in isoelectric-focusing buffer. Add 2%ampholytes between pH ranges of 3 to 10 and 4 to 6 in a ratio of one to four. Now fractionate the solubilized proteins in a liquid isoelectric-focusing system maintained at four degrees Celsius with a cooling water bath.

Conduct isoelectric-focusing separation following the manufacturer's instructions by applying a constant power of 12 watts. Collect the individual isoelectric-focusing fractions with a vacuum pump. Determine the pH values of each fraction.

Subject 50 micrograms of the separated fractions to SDS-PAGE and visualize the proteins using Coomassie Brilliant Blue or silver staining. Start by mixing the IEF-separated cell wall and cell membrane fractions with SDS gel electrophoresis sample buffer. Heat the mixture at 95 degrees Celsius for five minutes.

Separate the protein fractions in the second dimension, using 16 by 20 centimeter polyacrylamide gels with a 12.5%resolving gel and a 4%stacking gel. Then use a single 13-centimeter-long sample well to load the sample onto the gel. Perform electrophoresis at a constant current of 50 milliamperes per gel until the dye front reaches the bottom of the gel.

After electrophoresis, equilibrate the gel in elution buffer for 10 minutes. Transfer the gel to a whole gel eluter apparatus as per the instrument manufacturer's instructions. Run the eluter at a constant current of 250 milliamperes for one hour to elute the proteins from the gel, then use a vacuum pump to collect approximately 30 protein fractions, each measuring three milliliters, from the gel.

Quantify the protein concentration in the eluted fractions using the bicinchoninic acid protein assay. Subject 10 micrograms of the eluted protein fractions to SDS-PAGE analysis and visualize the bands by Coomassie Brilliant Blue staining. Distinct protein banding patterns were observed in the first supernatant:cell wall, cell membrane, and cytosol fractions of Mycobacterium tuberculosis, confirming successful subcellular fractionation.

The isoelectric focusing of cell wall proteins showed the highest protein concentration in fractions with pH below 2.5. SDS-PAGE of the cell wall IEF fractions revealed distinct protein bands, predominantly in Fractions 1 to 8. Cell membrane protein separation by IEF revealed major protein concentration peaks between pH 4 and pH 10, with the highest concentration occurring around pH 10.

SDS-PAGE of the cell membrane IEF fractions showed diverse protein profiles with clearer bands in Fractions 1 to 9. Preparative SDS-PAGE separation of the IEF-resolved cell wall proteins produced fractions predominantly under 100 micrograms per milliliter. SDS-PAGE of the eluted cell wall protein fractions displayed a wide range of protein molecular weights, with more concentrated bands in Fractions 6 to 10.

Preparative SDS-PAGE of cell membrane protein fractions revealed that over half of the fractions had protein concentrations between 50 and 199 micrograms per milliliter. SDS-PAGE of the eluted cell membrane protein fractions revealed concentrated bands in Lanes 1 to 8 and 13 to 16.