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

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 recognized to initiate an innate immune response, such as pathogen-associated molecular patterns (PAMPs) by immune cells, thereby initiating an immune response. For example, Mycobacterial lipoproteins such as LprG, LpqH, LprA, and PhoS1 are recognized by Toll-like receptor 2³.

The hydrophobic nature of cell wall and membrane proteins presents significant technical challenges in terms of their solubilization and separation. Studies have shown that membrane proteins constitute approximately 20% to 30% of the coding regions of all organisms, including mycobacteria^4,5. Due to their intrinsic nature and technical challenges, cell wall and membrane proteins are more difficult to solubilize and isolate compared to soluble proteins. As a result, only 2% of membrane protein structures have been reported⁶. Several factors contribute to the difficulty of isolating these proteins. The primary challenge is their low abundance within the cell, which complicates the isolation of individual proteins for further characterization⁷. Additionally, these proteins tend to aggregate, further complicating their solubility. This issue can be addressed by adding detergents to aid in solubilization. However, some proteins may still form aggregates even in the presence of detergents, in which case chaotropic agents and reducing agents can be used to improve solubility.

The use of detergents for solubilization can interfere with subsequent separation methods. For instance, charged detergents cannot be used with ion exchange chromatography, and all detergents can disrupt hydrophobic interaction chromatography⁸. Therefore, selecting a detergent compatible with the chosen separation method is critical. Another important step in cell wall and membrane protein isolation is the removal of detergents used for solubilization, as these can interfere with subsequent functional analyses of the proteins.

We have developed a robust methodology that overcomes many of the challenges associated with the analysis of membrane proteins, particularly the hydrophobic proteins from the cell wall and cell membrane. A key challenge in studying these proteins is their solubilization, as their hydrophobic nature makes them difficult to extract and analyze. The methodology described here employs a specially formulated isoelectric focusing (IEF) buffer, which efficiently solubilizes cell wall and cell membrane proteins. This solubilization step is critical as it enables the subsequent analysis of these hydrophobic proteins.

An additional advantage of this method is its ability to reduce the complexity of the protein mixture. By employing two complementary separation techniques-based on the isoelectric point (pI) and molecular weight of proteins-this methodology efficiently separates cell wall and cell membrane proteins into simplified fractions. Each fraction typically contains only one to three proteins, significantly enhancing the resolution and facilitating downstream characterization.

This methodology, developed using Mycobacterium tuberculosis cell wall and cell membrane proteins as a model system, effectively addresses the challenges associated with isolating and analyzing hydrophobic proteins. It provides a reliable approach for the isolation, separation, and characterization of these complex protein fractions, advancing the ability to study them in detail.

Protocol

This study has complied with the relevant governmental guidelines and was approved by the Institutional Scientific Advisory Committee and Ethical Committee. Informed consent was obtained from the study population before the samples were drawn.

1. Culturing of M. tuberculosis

1. Transfer approximately 10 M. tuberculosis colonies from Lowenstein-Jensen slants to 2 mL of 7H9 broth medium (suspend 2.35 g of Middlebrook 7H9 Broth Base in 400 mL of distilled water, 50 mL of Oleic Albumin Dextrose Catalase [OADC] supplement, 0.5 g of Tween 80, and make up to 500 mL)
   NOTE: M. tuberculosis is a Risk Group 3 pathogen; it should be handled in a biological safety cabinet (BSC) in the BSL3 facility. Appropriate personal protective equipment (PPE) must be used.
2. Disperse the cells using glass beads under sterile conditions in a bottle shaker at 15 rpm for 1 min.
3. Transfer the bacterial suspension to 10 mL of 7H9 broth medium in a McCartney bottle and incubate at 37 °C for 2 weeks in a shaker incubator at 30 rpm.
4. Scale up the log-phase culture to 200 mL of 7H9 broth medium and grow in a shaker culture for 4 weeks at 30 rpm.
5. Further, scale up the culture in a 1-L flask containing 500 mL of 7H9 broth medium and grow as a shaker culture at 30 rpm for 4 weeks at 37 °C.
6. Harvest the mycobacteria by centrifuging at 1741 g for 30 min.

2. Preparation of whole cell lysate proteins

1. Subject the isolated bacterial pellet to sonication following a standard protocol.
2. Prepare the breaking buffer with the following composition: 20 mM Tris with 8.5% (8.5 g per 100 mL) NaCl.
3. Adjust the final pH to 7.4 with concentrated HCl.
4. Add freshly prepared protease inhibitors to the breaking buffer with the following concentrations (details of protease inhibitors to be added are listed in Supplementary Table 1)
5. Treat M. tuberculosis bacilli (100 mL packed volume and weighed) for every 2 g of the bacterial cell pellet, add 1 mL of breaking buffer with lysozyme (1 mg/5 mL of cell suspension) and incubate for 15 min at 37 °C with gentle stirring.
6. Suspend the bacilli in twice its volume of breaking buffer.
7. Add 0.04% (wt/vol) sodium azide and 0.05% Tween 80 to the buffer.
8. Disrupt the bacterial cells in the breaking buffer using a sonicator with a cycle of 9 s on and 9 s off for 30 min at 40% amplitude, keeping the suspension on ice to prevent protein denaturation.
9. Centrifuge the prepared lysate at 3438 g for 15 min at 4 °C to remove unbroken cells.
10. Concentrate the supernatant using centrifugal concentrators (3 kDa cutoff- 5 mL).
11. Determine the protein concentration in the supernatant using the bicinchoninic acid (BCA) protein quantification assay using BSA protein as standard.
    NOTE: In SDS-PAGE analysis, load 50 µg protein for analysis.

3. Isolation of cell wall and cell membrane proteins by ultracentrifugation

1. Isolate cell wall, cell membrane, and cytosolic proteins from the whole cell lysate following the protocol by Raja et al.⁹. as described below.
2. Centrifuge the whole cell lysate at 13751 × g at 4 °C for 30 min, separating the cell wall proteins as a pellet.
3. Subject the supernatant to ultracentrifugation at 100,000 × g at 4 °C for 4 h using an ultracentrifuge, separating the cell membrane proteins as a pellet and leaving the cytosolic proteins in the supernatant.
4. Estimate protein concentrations in the cell wall and cell membrane fractions using the BCA protein assay.
   NOTE: In SDS-PAGE analysis, load 50 µg protein for analysis. Separated cell wall and cell membrane are stored in a -80 °C freezer till further analysis.

4. Preparative liquid-phase isoelectric focusing (Rotofor [IEF apparatus])

1. Solubilize the cell wall and cell membrane proteins isolated by ultracentrifugation in an IEF buffer containing 8 M urea, 1 mM DTT, 5% glycerol, 2% digitonin.
2. Add 2% ampholytes (pH 3.0 to 10.0 and pH 4.0 to 6.0 at a 1:4 ratio)
3. Fractionate the solubilized proteins using a liquid IEF system maintained at 4 °C with a cooling water bath.
4. Conduct the IEF separation according to the manufacturer's instructions, applying a constant power of 12 W until the voltage stabilizes (between 1300 V and 1600 V).
5. Continue the IEF run for an additional 30 min after voltage stabilization, with a total run time of approximately 5-6 h.
6. Collect the individual IEF fractions using a vacuum pump and determine their pH values.
7. Subject the separated fractions (50 µg) to SDS-PAGE and visualize the proteins using Coomassie Brilliant Blue (CBB) or silver staining. Store the separated IEF fractions in a -80 °C freezer till further analysis.

5. Preparative SDS-PAGE and whole gel elution

1. Mix the IEF-separated cell wall and cell membrane fractions with 6x SDS-PAGE sample buffer and heat at 95 °C for 5 min before SDS-PAGE analysis.
2. Separate the protein fractions in the second dimension using 16 cm 20 cm polyacrylamide gels consisting of a 12.5% resolving gel and a 4% stacking gel.
3. Use a single 13-cm-long sample well for loading the sample.
4. Conduct electrophoresis at a constant current of 50 mA/gel until the dye front reaches the bottom of the gel.
5. Equilibrate the gel in elution buffer (60 mM Tris, pH 9.4; 40 mM 3-(Cyclohexylamino)-1-propanesulfonic acid [CAPS]) for 10 min after the electrophoresis run.
6. Transfer the gel to a whole gel eluter apparatus as described by the instrument manufacturer.
7. Elute the proteins from the gel by running the eluter at a constant current of 250 mA for 1 h.
8. Collect approximately 30 protein fractions, each with a volume of 3 mL, from each gel using a vacuum pump.
9. Quantify the protein concentration in the eluted fractions using the BCA protein assay.
10. Subject 10 µg of the eluted fractions to SDS-PAGE analysis and visualize by Coomassie brilliant blue (CBB) staining.
    NOTE: Separated whole gel eluted fractions were stored in the -80 °C freezer till further analysis.

Results

Isolation of cell wall and cell membrane proteins by high-speed ultracentrifugation
Cell wall and membrane proteins were isolated from whole cell lysates via ultracentrifugation as outlined in the protocol. SDS-PAGE analysis of the separated subcellular fractions-cell wall, cell membrane, and cytosol protein profiles of M. tuberculosis is shown in Figure 1.

Separation of cell wall proteins by preparative liquid-phase isoelectr...

Discussion

The hydrophobic nature of cell wall and membrane proteins presents significant experimental challenges in their solubilization and separation. In the abovementioned protocol, we described a novel two-dimensional electrophoresis-based separation method to effectively isolate these hydrophobic proteins. In this protocol, we initially separated the membrane and cell wall proteins based on their isoelectric points using the liquid IEF system. To solve the solubilization problem, we used a high concentration of chaotropic age...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7H9 broth	Himedia	M198
40% Bio-Lyte 3/10 Ampholyte	BIO-RAD	1631113
BCA protein assay	MERCK	BCA1
Bio-Lyte 5/7 Ampholyte	BIO-RAD	1631152
centrifugal concentrators 3 kDa	MERCK	Z629456
Digitonin.	MERCK	D141
Dnase	MERCK	11284932001
DTT	MERCK	D9779
EDTA	MERCK	E9884
EGTA	MERCK	E3889
Glycerol	MERCK	G5516
Leupeptin	MERCK	E18
Pepstatin A	MERCK	P5318
PMSF	MERCK	78830
Rnase	MERCK	10109134001
Rotofor	BIO-RAD	170-2986	isoelectric focusing (IEF) apparatus
Sonicator	Vibra-Cell, Sonics	VC 750
TLCK	MERCK	T7254
TPCK	MERCK	4376
urea	MERCK	U5378
Whole Gel Eluter apparatus	BIO-RAD	1651250