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

This study aims to separate the culture filtrate proteins of mycobacterium tuberculosis to find out the key protein fractions involved in immune response and disease, which may help in developing better diagnostics or vaccines. We are trying to find out which fractions are the most immunogenic and how they can contribute to the bacteria's ability to cause and sustain infection. Recent advancement in the electrophoresis technique now enable us to separate the highly complex proteomes into simple fractions, such as cationic two-to-four proteins, allowing precise downstate proteomic and immunological analysis.

One major expanded challenges in proteomics is the limited loading capacity of during electrophoresis. Loading too much of protein, say, about 350 milligram of protein, or more can lead to precipitation, affecting the quality of separation. Additionally, the electrophoretic process can denature the protein, making them unsuitable for further functional studies or assays that require proteins in their native state.

Using our protein suppression method, we successfully identified 27 new T-cell antigens specific to Mycobacterium tuberculosis. These antigens show strong promise for the development of more accurate diagnostic tools and next-generation TB vaccines. This protocol addresses the gap into resolving complex proteins by allowing separation into well-defined and simplified protein fractions.

These fractions can be directly used for proteomic and immunological studies, helping in faster identification of disease-specific biomarkers without the need for further purification. To begin, prepare Sauton's liquid medium by combining the given reagents in 950 milliliters of distilled water. Make up the volume to one liter and autoclave at 15 PSI for 20 minutes.

Now, transfer colonies of Mycobacterium tuberculosis grown on Lowenstein-Jensen, or LJ slopes, into two milliliters of Sauton's liquid medium. Using glass beads, vigorously shake the suspension under sterile conditions to break up mycobacterial clumps. To initiate mycobacterial growth, transfer the bacterial suspension into a McCartney bottle containing 10 milliliters of Sauton's liquid medium.

Incubate the bottle at 37 degrees Celsius for two weeks. Scale up the culture by transferring the grown culture to 200 milliliters of Sauton's medium in a one liter bottle. Place the bottle on a shaker and incubate at 37 degrees Celsius for four weeks.

Transfer the log phase culture into a four liter flask containing two liters of Sauton's medium. Grow the culture at 37 degrees Celsius under stationary conditions for an additional four weeks until a surface pellicle forms. Transfer the culture into centrifuge tubes and harvest them by centrifuging at 1, 000 to 1, 500 G for 30 minutes at room temperature.

Filter the supernatant through a 0.45 micrometer vacuum filtration system. Then, using a tangential flow filtration system with a 10 kilodalton cutoff, concentrate the filtrate containing culture filtrate proteins. Estimate the protein content using a commercially available bicinchoninic acid assay kit.

Aliquot the proteins, add sodium azide to a final concentration of 0.2%and store at minus 80 degrees Celsius. Solubilize mycobacterium tuberculosis culture filtrate protein in 60 milliliters of isoelectric focusing, or IEF separation buffer, containing eight molar urea, one millimolar dithiothreitol, 5%glycerol, 2%digitonin, and 2%ampholytes. Fill the anode chamber with 0.1 molar phosphoric acid and the cathode chamber with 0.1 molar sodium hydroxide.

Assemble the preparative isoelectric focusing cell according to the manufacturer's instructions. Load up to 350 milligrams of protein onto the IEF system. Focus the proteins at four degrees Celsius using a cooling circulatory water system.

Apply a constant power of 12 watts until the voltage stabilizes at approximately 1, 400 volts before continuing for an additional 30 minutes. Harvest the IEF fractions and determine their pH. Analyze each aliquot by SDS-PAGE.

Prepare samples for SDS-PAGE by mixing the IEF fractions with a commercially available 6X SDS-PAGE sample buffer. Heat the mixture at 95 degrees Celsius for five minutes. Load up to 10 milligrams of protein into a 16 by 20 centimeter polyacrylamide gel with a 4%stacking gel and a 12.5%resolving gel.

Run the electrophoresis at a constant current of 50 milliamperes until the dye front is two centimeters from the bottom. Now, equilibrate the gel in an elution buffer for 10 minutes. Transfer the gel to the whole gel eluter instrument.

Elute proteins by applying a constant current of 250 milliampere for one hour. Next, collect approximately 30 fractions of 2.5 milliliters each. Determine protein concentration using the bicinchoninic acid method, following the manufacturer's instructions.

Analyze 10 micrograms of protein from each fraction by SDS-PAGE. This image displays a two dimensional SDS-PAGE analysis, where 100 micrograms of culture filtrate proteins produced nearly 100 distinct protein spots, indicating a highly complex protein composition. SDS-PAGE analysis of three representative fractions from different separation runs showed distinct banding patterns, confirming variability in protein composition across the samples.

The pH values of the separated fractions gradually increased from 2.54 to 12.9, with the majority of proteins concentrated in fractions with pH between four and six. SDS-PAGE analysis of whole gel eluted isoelectric focusing fraction 12 revealed that each fraction contained one to three protein bands, confirming the successful separation and resolution of proteins into distinct bands.