This article describes a detailed protocol for the purification of a polyhistidine-tagged protein from Streptococcus mutans cultures. The process involves ammonium sulfate precipitation, dialysis, and immobilized metal affinity chromatography (IMAC) to achieve high purity of the target protein.
Take a culture of Streptococcus mutans expressing a polyhistidine-tagged protein secreted into the medium.
Centrifuge the culture and collect the protein-rich supernatant.
Add ammonium sulfate and incubate with stirring to precipitate proteins by reducing their solubility.
Transfer the solution to a tube, centrifuge, and discard the supernatant.
Resuspend the pellet in binding buffer containing salts and low imidazole to stabilize the protein.
Dialyze the solution against fresh binding buffer to remove excess salts.
Centrifuge the dialyzed solution to pellet insoluble debris, then filter the supernatant.
Mix the filtrate with nickel-charged affinity resin and incubate to allow selective binding of the polyhistidine-tagged protein to the nickel ions.
Load the mixture onto the chromatography column and wash with binding buffer to remove unbound proteins.
Next, add an elution buffer with high imidazole to displace histidine from nickel ions and release the polyhistidine-tagged protein.
Collect this eluate containing the purified proteins.
Centrifuge the bacterial culture suspension for 20 minutes at 10,000 times g at four degrees Celsius.
Transfer the culture supernatant into a three-liter glass beaker. To concentrate the proteins from the culture supernatant, place the two-liter supernatant on a magnetic stirrer and start vigorous stirring. Add 1,122 grams of ammonium sulfate and allow the precipitate to form with vigorous stirring at four degrees Celsius over a 4 hour period or overnight.
Next, centrifuge the ammonium sulfate precipitated solution at 15,000 times g for 20 minutes at four degrees Celsius. Decant the supernatant. With a spatula, collect the precipitate and transfer it into a 200-milliliter glass beaker.
Resuspend the pellets in 35 milliliters of binding buffer. Then, transfer each approximately 25 milliliters of the suspension into a regenerated cellulose dialysis tubing. Place the dialysis tubing into 2.5 liters of the stirring binding buffer on a stirrer at four degrees Celsius to dialyze the suspension.
Replace the dialysis solution after two hours and continue dialysis overnight. Next, transfer the dialyzed suspension from the tubing to centrifuge tubes and place the tubes in the centrifuge at 20,000 times g for 10 minutes at four degrees Celsius. With suction filtration equipment, pour the supernatant over a 0.2-micrometer membrane filter to filter.
Transfer the filtrate to a 75-milliliter flask.
To fractionate the polyhistidine-tagged gtfSI from the filtrated suspension, first prepare an immobilized metal affinity chromatography. After equilibrating the resin according to the manuscript, close the Hoffman pinch cock.
Add five milliliters of the filtered suspension into the column to make a slurry. Then, transfer all the slurry to the remaining filtered suspension and swirl the mixture gently for 30 minutes at four degrees Celsius. Load the mixture back into the column.
Open the Hoffman pinch cock to remove the suspension by the gravity flow. Wash the IMAC resin with 20 milliliters of binding buffer and then, elute 20 milliliters of elution buffer to obtain the recombinant gtfSI.
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