One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

This research investigates a simpler methodology for extracting and characterizing active gelatinases from Leptospira, utilizing zymography to confirm their enzymatic activity, and addressing challenges in isolating membrane proteins from bacteria. Current technologies include mass spectrometry-based proteomics, two-dimensional gel electrophoresis, bioinformatic tools, zymography, and LC-MS/MS, enabling detailed protein profiling, functional analysis, and identification of virulence factors in Leptospira, and vaccine candidate discovery. Characterizing active outer membrane proteins such as Leptospira gelatinases poses a challenge, particularly due to the membrane localization, which requires efficient disruption and solubilization methods to maintain the protein's integrity and accuracy.

This protocol addresses the problem of extracting active outer membrane gelatinases from Leptospira by providing a one-stop extraction process for use in pathogenesis investigation. Our laboratory focuses on leveraging advanced high-resolution LC-MS/MS to systematically identify outer membrane and secretory proteins, particularly focusing on proteinases involved in the extracellular matrix degradation. By elucidating their roles in pathogenesis, we aim to identify sensitive and specific biomarkers for the early-stage detection of leptospirosis.

To begin, grow the organism in Ellinghausen-McCullough-Johnson-Harris medium containing 10 milligrams per milliliter of BSA at 30 degrees Celsius. Take 10 milliliters of Leptospira culture in the mid-logarithmic phase of growth, containing 3.5 times 10 to the power of 8 cells per milliliter for proteinase extraction. Centrifuge the culture at 6, 000G for 10 minutes.

Discard the supernatant into a container for decontamination. Suspend the pellet in one milliliter of wash buffer, and transfer it to a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 6, 000G for five minutes, and repeat the wash once more.

Now, add 100 microliters of extraction buffer to the Leptospira pellet, and vortex for five minutes. Incubate the mixture at four degrees Celsius for one hour. Then, centrifuge the suspension at 15, 000G at four degrees Celsius for 10 minutes.

Post-centrifugation, collect the extracted protein. Prepare the resolving gel mix for sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized gelatin. Pour the resolving gel mix into the casting chamber up to approximately three to five millimeters below the mark for the bottom of the wells.

Slowly pour water to form an even one millimeter layer over the gel, and remove any bubbles, and let the gel set for one hour. Before preparing the stacking gel, tilt the setup to decant the water from the top of the set resolving gel, and blot with a narrow strip of filter paper. Add 0.03 milliliters of 10%ammonium persulfate to the stacking gel mix, and stir gently to avoid bubble formation.

Now, pour the stacking gel mix onto the resolving gel and immediately place the comb without trapping air bubbles. Next, fix the gel into the electrophoresis apparatus and remove the lower spacer. After placing the gel in the tank, add an equal volume of sample buffer to the protein extract.

Prepare a positive control with two microliters of human serum with an equal volume of sample buffer. Incubate the samples at 37 degrees Celsius for 30 minutes to allow SDS to bind to the protein. Now, carefully load the prepared samples and control into the gel wells using a narrow-tipped pipette.

Start the electrophoresis at six milliamperes per gel until the dye front enters the resolving gel. Then increase the current to 12 milliamperes per gel, and continue running until the dye front reaches one centimeter above the bottom of the gel. After electrophoresis, carefully dismantle the glass plates using a plate separation tool or plastic spatula.

Mark the orientation of sample loading by cutting the right lower corner of the gel, and then mark the pre-stained marker bands. Submerge the gel in a renaturation solution containing Triton X-100 in a gel tray, and incubate at room temperature for 30 minutes without shaking. Replace the renaturation solution with activation buffer, and incubate again for 18 hours.

After removing the activation buffer, rinse the gel with water for one minute, and add the staining solution, ensuring the gel is fully submerged. Gently shake the gel at low speed until the gel turns completely blue. Now, rinse the gel surface with water for one minute to remove excess stain, and add the de-staining solution.

Replace the solution several times until the desired contrast is achieved. Finally, scan the gel using a gel documentation system or document scanner to compare the protein bands with the standard markers. The zymogram of Leptospira protein and gelatinases from human serum shows proteases as white clear bands against a blue background.

The bands represent areas where the copolymerized gelatin is degraded, and hence, not stained, while other areas of the entire gel are stained blue due to the presence of gelatin.