A Procedure for the Purification of a Polyhistidine-Tagged Protein from Streptococcus mutans

Samenvatting

Source: Murata, T., et al. Purification of a High Molecular Mass Protein in Streptococcus mutans. J. Vis. Exp. (2019)

This video demonstrates a step-by-step procedure for the purification of a polyhistidine-tagged protein secreted from Streptococcus mutans.

Protocol

1. Concentrate proteins from the culture supernatant by ammonium sulfate precipitation.
   NOTE: Extract from cell bodies if a target protein is intracellular.
   1. Centrifuge the bacterial culture (Streptococcus mutans) suspension for 20 min at 10,000 x g at 4 °C. Recover the culture supernatant into a 3 L glass beaker.
   2. Add 1,122 g of ammonium sulfate to 2 L of the supernatant (80% saturation) with vigorous stirring using a magnetic stirrer. Allow the precipitate to form for 4 h or more at 4 °C with stirring.
      NOTE: The precipitate formation can be continued overnight.
   3. Centrifuge the ammonium sulfate-precipitated solution at 15,000 x g for 20 min at 4 °C. Decant the supernatant.
   4. Collect the precipitate with a spatula and transfer into a 200 mL glass beaker. Resuspend the pellets in 35 mL of binding buffer (50 mM NaH₂PO₄, 300 mM NaCl, 5 mM imidazole, pH 8.0).
   5. Dialyze the suspension against 2,500 mL of the binding buffer using a regenerated cellulose dialysis tubing, at 4 °C, with stirring. Replace the dialysis solution after 2 h and continue dialysis overnight.
   6. Replace the dialysis solution again. Continue to dialyze for an additional 2 h.
2. Centrifuge the dialyzed suspension at 20,000 x g for 10 min at 4 °C. Filter the supernatant through a membrane filter using suction filtration equipment. Transfer the filtrate to a 75 cm² flask.
3. Fractionate the polyhistidine-tagged Glucosyltransferase-SI (GTF-SI) from the filtrated suspension by immobilized metal affinity chromatography (IMAC).
   1. Transfer 2 mL of the Ni-charged IMAC resin slurry (approximately 1 mL of resin) to a chromatographic column whose outlet is fitted to a silicone tube. Remove the storage solution by the gravity flow.
      CAUTION: Do not allow the resin to dry out throughout the IMAC.
   2. Add 3 mL of distilled water into the column to wash the resin. Add 5 mL of the binding buffer to equilibrate the resin.
   3. Shut off the flow with a Hoffmann pinch cock. Add 5 mL of the filtered suspension from step 2 to make the slurry.
   4. Add all the slurry to the remaining filtered suspension. Swirl the mixture gently for 30 min at 4 °C.
   5. Load the mixture back on the column. Remove the suspension by the gravity flow. Wash the IMAC resin with 20 mL of binding buffer.
      NOTE: Adjust the flow rate to approximately 2 mL/min with a Hoffmann pinch cock throughout the subsequent IMAC.
   6. Elute the recombinant GTF-SI with 20 mL of the elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 500 mM imidazole, pH 8.0).
      NOTE: The protocol can be paused here. The eluate should be stored at 4 °C. IMAC resin can be reused.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Centrifugal ultrafiltration unit	Sartorius	VS2032	Buffer replacement and protein concentration
Centrifuge	Kubota	7780II
Chromatographic column	Bio-Rad	7321010	For IMAC
Dialysis membrane clamp	Fisher brand	21-153-100
Dialysis tubing	As One	2-316-06
Imidazole	Wako	095-00015	Binding buffer and elution buffer preparation
Incubator	Nippon Medical & Chemical Instruments	LH-100-RDS	Temperature setting: 37 °C
Microcentrifuge	Kubota	3740
Sodim chloride	Wako	191-01665	Preparation of binding buffer and elution buffer
Sodium dihydrogen phosphate dihydrate	Wako	192-02815	Preparation of binding buffer and elution buffer
Sodium hydroxide	Wako	198-13765	Preparation of binding buffer and elution buffer
Ammonium sulfate	Wako	015-06737	Ammonium sulfate precipitation
Ni-charged resin	Bio-Rad	1560133	For IMAC
Membrane filter	Merck Millipore	JGWP04700	0.2 µm diameter