The overall goal of this protocol is to facilitate the isolation and purification of S100A12 in its active form so that it can be utilized for antimicrobial assays. This method can help answer key questions in the microbiology field, such as what is the role of S100A12, an antimicrobial peptide, which is produced in response to infection by pathogenic bacteria. The main advantage of this technique is that our optimized protocol provides high yields of stable pure S100A12 dimer, which has the capacity to bind and sequester nutrient metals.
Generally, individuals new to this method will struggle because S100A12 forms oligomers of varying sizes under certain conditions, and these oligomers have diminished antimicrobial activity in vitro. We first had the idea for this method when we began purifying and studying the antimicrobial activity of a related S100A family protein called calprotectin. Visual demonstration of this method is critical to the size and column final purification steps.
And the antimicrobial assays are important to the success of this process. To being this procedure, transform competent BL21 DE3 cells as outlined in the text protocol. Next, add one to five microliters of plasmid to 50 microliters of bacteria in a micro centrifuge tube on ice.
Incubate for 20 minutes. After this, heat-shock the cells at 42 degrees Celsius for 30 seconds. Incubate on ice for two minutes.
Then, add 500 microliters of SOC media. Incubate at 37 degrees Celsius on an orbital shaker at 250 rpm for one hour. Next, plate 150 microliters of the transformation reaction on LB arga medium, supplemented with 100 micrograms per milliliter ampicillin.
Incubate for 12 to 16 hours at 37 degrees Celsius. After incubation, pick a single colony and inoculate two milliliters of ampicillin supplemented LB medium. Incubate at 37 degrees Celsius on an orbital shaker, at 300 rpm, for four to six hours.
Next, add 500 microliters of starter culture to 50 milliliters of ampicillin supplemented ZYM 5052 autoinduction media. Shake at 300 rpm for 24 hours at 37 degrees Celsius. Then, transfer the bacteria suspension to a centrifuge tube.
Centrifuge at 4, 000 times G and four degrees Celsius for 10 minutes. After centrifugation, decamp the media, log the sample, and store the cell paste at minus 80 degrees Celsius. To begin, resuspend the cell paste in 30 milliliters of 20 millimolar of tris, at pH 8.0 Next, sonicate the suspension on ice at 20 watts using a five seconds on and five seconds off cycle for five minutes to lyse the cells.
Transfer the sonicated solution to high-end centrifugation tubes. Centrifuge at 20, 000 times G at four degrees Celsius for 30 minutes to clarify the cell lysate. After this, decant the supernatant into a clean 100 milliliter polypropylene beaker.
Place the beaker on ice to cool the solution. Add a stir bar and slowly add 11.20 grams of ammonium sulfate. Stir the solution on ice for one hour.
Then, transfer the solution to centrifuge tubes and centrifuge at 20, 000 times G at four degrees Celsius for 20 minutes to pellet the precipitated protein. Decant the supernatant into dialysis tubing. Dialyze against one liter of 20 millimolar tris at four degrees Celsius and pH 8.0 Change the dialysis buffer twice with four hours between changes.
After this, equilibrate a five milliliter sepharose column with 10 milliliters of 20 millimolar tris. Using the sample pump, load approximately 40 milliliters of S100A12 solution. Collect the flow through.
Next, wash the column with 20 millimolar tris. Develop the column using a zero to 30%gradient over 19 column volumes. Collect five milliliter fractions.
Use MES SDS page with coomassie staining to analyze a 10 microliter aliquot of each fraction. Then, pull the fractions containing S100A12. Using an ultra filtration device, concentrate the fractions to five milliliters.
After this, equilibrate an S75 column, with a 120 milliliter solution of 20 millimolar tris and 100 millimolar sodium chloride. Inject the five milliliter sample of concentrated fractions. Next, develop the column at a flow rate of one milliliter per minute over 120 milliliters.
Collect five milliliter fractions. Using SDS-PAGE with coomassie staining, analyze a 20 microliter aliquot of each sample. Then validate the protein identity and pull the fractions as outlined in the text protocol.
To begin, streak H.pylori into brucella broth supplemented with 1X cholesterol. Culture overnight at 37 degrees Celsius with shaking at 250 rpm with air supplemented with 5%carbon dioxide. Dilute the H.pylori one to ten to a mixture of 50%brucella broth and 50%calprotectin buffer plus 1X cholesterol.
And add S100A12 protein and/or exogenous metals as necessary. The antimicrobial assays are a critical point. It is important to use calprotectin buffer to ensure the stability of the S100A12 dimers, and to provide a metal deficient mineral medium for the bacteriological growth assays so that the protein will not be overwhelmed with excess micronutrients, which are commonly present in rich complex bacteriological media.
The following day, serially dilute the samples. Next, plate onto blood agar plates. Allow the bacteria colonies to grow for two to three days at 37 degrees Celsius in air supplemented with 5%carbon dioxide.
After this, calculate bacterial growth as outlined in the text protocol. In this study, a refined method is presented for expressing and purifying S100A12 in its active metal-binding configuration. The three-step purification process begins with an ammonium sulfate precipitation of endogenous E.coli proteins, which is followed by anion-exchange chromatography.
After tracking the protein via an SDS-PAGE with coomassie brilliant blue. The fractions containing S100A12 are pulled for size exclusion chromatography. S100A12 is a homodimer with 92 amino acids per subunit and has a total molecular weight of about 21 kilodaltons.
The antimicrobial activity of S100A12 is then investigated via quantitative microbiological culture techniques. Enumeration of bacteria cells reveals that exposure to 100 micrograms per milliliter of S100A12 in the presence or the absence of an exogenous source of nutrients zinc did not significantly inhibit bacteria viability. However, exposure to 1, 000 micrograms per milliliter of S100A12 in medium alone results in a 69-fold decrease in bacterial viability.
Adding an exogenous source of nutrient zinc reverses this result, which demonstrates that the antimicrobial activity of S100A12 is dependent upon its zinc-sequestration activity. Once mastered, the protein expression in purification can be done in three to four days, if it is performed properly. Following this procedure, other methods like inductively coupled mass spectrometry can be used in order to answer additional questions.
Like how does metal sequestration restrict microbial growth? After its development, this technique paved the way for researchers in the field of microbiology to explore nutritional immunity of H.pylori. After watching this video, you should have a good understanding of how to express, purify, and quantify the activity of the antimicrobial protein S100A12.
Don't forget that working with H.pylori can be extremely hazardous. And precautions, such as working in a BS2 certified space should always be taken when performing this procedure. The implications of this technique extend toward gastric diseases, including ulcer, gastritis, and invasive adnexal carcinoma.
By understanding these processes, we will be better equipped to build novel antimicrobial molecules that target these processes. Though this method can provide insight into the biochemistry of S100A family proteins, it can also be applied to studying their role in immune signaling and numerous disease processing such as cancer, arthritis, and infectious disease.
