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

Abstract

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. Cen....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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