Separation and Fractionation of Culture Filtrate Proteins (CFPs) from Mycobacterium tuberculosis

Summary

This protocol describes the culturing, harvesting, and concentration of secreted proteins of Mycobacterium tuberculosis. Culture filtrate proteins (CFPs) are quantified, separated into 20 fractions by isoelectric focusing, and further resolved into 30 fractions using a preparative SDS-PAGE and an electroelution system.

Abstract

Secreted proteins of Mycobacterium tuberculosis (M. tuberculosis) play a crucial role in tuberculosis pathogenesis, immune modulation, and disease progression. Understanding their composition and function is essential for identifying novel biomarkers that can aid in tuberculosis diagnosis, vaccine development, and therapeutic interventions. This study focuses on the systematic isolation, fractionation, and characterization of culture filtrate proteins (CFPs) to enable comprehensive immunological and proteomic analyses. CFPs were obtained from M. tuberculosis H37Rv cultures grown in chemically defined conditions to ensure controlled protein expression. The culture supernatant was purified through filtration and concentrated using a hollow fiber system to retain proteins above 10 kDa. Fractionation was achieved through liquid-phase isoelectric focusing, separating proteins based on their isoelectric points into 20 distinct fractions. A further resolution was performed using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution, yielding 30 protein fractions. The fractionated proteins obtained through this approach provide a valuable resource for immunological profiling and mass spectrometry-based proteomic analyses. By systematically isolating and characterizing secreted proteins, this study contributes to the identification of tuberculosis-specific protein biomarkers, which could improve diagnostic accuracy and advance our understanding of M. tuberculosis pathogenesis.

Introduction

Extensive research has highlighted the significant role of secreted proteins in the growth and immune interactions of Mycobacterium tuberculosis¹. The culture filtrate of M. tuberculosis contains a complex array of protein and non-protein components, making it a valuable resource for understanding its biology and immunogenic properties. Early proteomic studies identified nearly 450 proteins in the culture filtrate², many of which play a critical role in eliciting immune responses against tuberculosis. Identifying these immunologically relevant antigens is essential for developing innovative diagnostics and vaccines for tuberculosis. However, progress in this area is challenged by the complexity of the immune response to M. tuberculosis infection and the technical limitations of biochemical purification methods used to isolate mycobacterial proteins.

Various separation techniques have been employed to resolve M. tuberculosis proteins, facilitating the detection of minor yet functionally significant proteins and expanding the scope of proteomic studies. An ideal separation method effectively resolves complex protein mixtures into a limited number of distinct fractions. Two widely used approaches for separating mycobacterial proteins are chromatography-based and electrophoresis-based methods^1,2.

Chromatographic techniques, including affinity, ion exchange, and reversed-phase chromatography, have been the cornerstone of protein fractionation for over a decade. These methods have been extensively used for separating M. tuberculosis proteins³. However, they are limited by factors such as low protein yields and interference from non-protein components, which can complicate subsequent immunological analyses. These drawbacks restrict the utility of chromatography for identifying immunologically relevant antigens.

To address these limitations, a two-dimensional electrophoresis-based separation method was adopted. In the first dimension, proteins are separated based on their isoelectric focusing (IEF) using a preparative IEF system, which segregates proteins according to their isoelectric points. In the second dimension, proteins are further resolved by molecular weight using preparative SDS-PAGE, followed by elution with a whole-gel eluter instrument.

This two-dimensional approach offers several advantages. It significantly reduces the complexity of culture filtrate proteins (CFP) by fractionating them into 600 distinct fractions. Additionally, the fractions are suitable for direct immunological analysis because the elution process does not introduce components that interfere with cellular immunological assays^4,5,6.

This protocol details the preparation and separation of culture filtrate proteins for immunological analysis⁶.

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Growth of Mycobacterium tuberculosis and preparation of culture filtrate proteins (CFPs)

1. Preparation of Sauton's liquid medium
   1. Combine the following components in 950 mL of distilled water: Glycerol, 30.0 mL; Potassium dihydrogen orthophosphate (KH₂PO₄), 0.5 g; Magnesium sulfate, 0.5 g; Citric acid, 2.0 g; Casein hydrolysate, 0.5 g; Ferric ammonium sulfate, 0.05 g; Sodium glutamate, 4.0 g.
   2. Adjust the pH to 7.2 using 40% potassium hydroxide (approximately 5 mL/L).
   3. Make up the volume to 1 L and autoclave at 15 psi for 20 min.
2. Preparation of mycobacterial suspension
   1. Transfer colonies of Mycobacterium tuberculosis H37Rv (ATCC 27294) grown on Lowenstein-Jensen (LJ) slopes into 2 mL of Sauton's liquid medium.
   2. Break up mycobacterial clumps by vigorous shaking with glass beads under sterile conditions.
3. Initiating mycobacterial growth
   1. Transfer the bacterial suspension into a McCartney bottle containing 10 mL of Sauton's liquid medium.
   2. Incubate the bottle at 37 °C for 2 weeks.
4. Scaling up the culture
   1. Transfer the grown culture to 200 mL of Sauton's medium in a 1 L bottle.
   2. Incubate on a shaker at 37 °C for 4 weeks.
   3. Transfer the log-phase culture to a 4 L flask containing 2 L of Sauton's medium.
   4. Grow the culture at 37 °C under stationary conditions for an additional 4 weeks until a surface pellicle forms.
5. Harvesting the culture filtrate proteins (CFPs)
   1. Harvest the culture by centrifugation at 1,000-1,500 x g for 30 min at room temperature.
   2. Filter the supernatant through a 0.45 µm vacuum filtration system.
   3. Concentrate the filtrate containing CFP using a tangential flow filtration system with a 10 kDa cutoff.
6. Storing the harvested CFP
   1. Estimate the protein content using the commercially available BCA assay kit.
   2. Aliquot the proteins, add sodium azide to a final concentration of 0.2%, and store at -80 °C.

2. Preparative liquid phase isoelectric focusing (IEF)

1. Preparation of protein sample
   1. Solubilize Mycobacterium tuberculosis CFP in 60 mL of IEF separation buffer containing 8 M of urea, 1 mM of dithiothreitol (DTT), 5% glycerol, 2% digitonin, 2% ampholytes (pH 3.0-10.0 and pH 4.0-6.0 at a 1:4 ratio).
2. Setting up the IEF system
   1. Fill the anode chamber with 0.1 M phosphoric acid and the cathode chamber with 0.1 M sodium hydroxide.
   2. Assemble the preparative IEF cell according to the manufacturer's instructions (see Table of Materials).
3. Performing IEF
   1. Load up to 350 mg of protein onto the IEF system.
   2. Focus proteins at 4 °C with a cooling circulatory water system.
   3. Apply a constant power of 12 W until the voltage stabilizes at approximately 1400 V, then continue for an additional 30 min.
4. Collecting and analyzing the fractions
   1. Harvest IEF fractions and determine their pH.
   2. Analyze each aliquot by SDS-PAGE and visualize proteins using silver staining.

3. Preparative SDS-PAGE and whole-gel elution

1. Preparing samples for SDS-PAGE
   1. Mix IEF fractions with commercially available 6x SDS-PAGE sample buffer.
   2. Heat the mixture at 95 °C for 5 min.
2. Running preparative SDS-PAGE
   1. Load up to 10 mg of protein into a 16 cm × 20 cm polyacrylamide gel with a 4% stacking gel and a 12.5% resolving gel.
   2. Run electrophoresis at a constant current of 50 mA until the dye front is 2 cm from the bottom.
3. Eluting proteins from the gel
   1. Equilibrate the gel in elution buffer (60 mM of Tris, 40 mM of CAPS, pH 9.4) for 10 min.
   2. Transfer the gel to the whole-gel eluter instrument.
   3. Elute proteins by applying a constant current of 250 mA for 1 h.
4. Collecting and analyzing the fractions
   1. Elute the separated proteins into 30 individual fractions, each with a volume of 2.5 mL.
   2. Determine protein concentration using the BCA protein estimation method following the manufacturer's instructions.
   3. Analyze 10 µg of protein from each fraction by SDS-PAGE and visualize using silver staining.

Results

Figure 1 illustrates the complexity of the culture filtrate proteins (CFP) prepared. Initially, 100 µg of CFP was subjected to 2D-SDS PAGE analysis to assess its complexity. This analysis revealed the presence of nearly 100 distinct protein spots in the CFP (Figure 1).

The 20 fractions collected from each run were analyzed by SDS-PAGE. Of these, three representative fractions from three different runs are shown in

Discussion

Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis represent a complex mixture of diverse protein components present at varying concentrations. Proteomic studies have identified up to 1,314 proteins within the CFP^4,5. Numerous investigations have highlighted the crucial role of CFPs in modulating immune responses. Specifically, CFPs play an indispensable role in both cell-mediated immunity, characterized by T cell responses^4...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ÄKTA flux tangential flow filtration system	CYTIVA	29038437
Ampholytes (pH 3.0 to 10.0 )	BIO-RAD	1632094
Ampholytes (pH 4.0 to 6.0 )	BIO-RAD	1631143
BCA protein assay	MERCK	71285-M
CAPS	MERCK	C2632
Casein hydrolysate	MERCK	65072-00-6
Citric acid	MERCK	C0759
Digitonin	MERCK	300410
DTT	MERCK	D9779
Ferric ammonium sulfate	HIMEDIA	GRM10944
Glycerol	MERCK	G5516
Glycerol	MERCK	252859
Lowenstein-Jensen slants	HIMEDIA	SL001
Magnesium sulfate	MERCK	M7506
Millipore Stericup Quick Release Vacuum Filtration System	MERCK	S2HVU11RE
Phosphoric acid	MERCK	695017
Potassium dihydrogen orthophosphate	MERCK	P0662
Potassium hydroxide	MERCK	484016
Preparative IEF Cell (Rotofor)	BIO-RAD	170-2986
Sodium azide	MERCK	S2002
Sodium glutamate	MERCK	49621
Sodium hydroxide	HIMEDIA	MB095
Tris	MERCK	252859
Urea	MERCK	U5128
Whole Gel Eluter apparatus	BIO-RAD	1651250