Purification of a High Molecular Mass Protein in Streptococcus mutans

Summary

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Abstract

Elucidation of a gene's function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3′ end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Introduction

Analysis of a gene's function usually involves comparison of phenotypic traits of wild-type strains to strains in which the gene of interest has been disrupted.Once the gene-disrupted strain is produced, exogenous addition of the gene product allows functional restoration.

The most common method for obtaining purified gene products required for subsequent restoration assays is by performing heterologous expression in Escherichia coli¹. However, the expression of membrane proteins or high molecular mass proteins is often difficult using this system¹. In these cases, the target prot....

Protocol

NOTE: Generation of S. mutans ∆gtfC, in which the entire coding region of the gtfC gene is replaced with spc^r, must be completed prior to performing these protocols. Refer to the published article for details on generation⁵.

1. Primer design

1. Prepare primers for the construction of S. mutans His-gtfC.
   NOTE: The primer sequences used in this protocol are shown in Table 1. Two-step fusion PCR method for the generation of S. mutans His-gtfC is sche....

Results

Figure 3 shows the size of each amplicon from the first PCR (Figure 3A) and second PCR (Figure 3B). The size of each amplicon corresponded with the predicted size, as described in Table 1. Figure 4A shows S. mutans colonies transformed with the second PCR product and plated on the BHI agar plates containing spectinomycin. Colony PCR products wer.......

Discussion

The design of primers is the most critical step in the protocol. The sequences of the gtfC-reverse and spc^r-forward primers were automatically determined based on the sequences of both the 3′ end region of gtfC and the 5′ end region of spc^r. Each primer includes 24 complementary bases that encode a GS linker and a His-tag-coding sequence at their 5' regions. Disruption of the native regulatory sequences located in the upstream flanking regions can .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Nippon Genetics	NE-AG02	For agarose gel electrophoresis
Anaeropack	Mitsubishi Gas Chemical	A-03	Anaerobic culture system
Anti-His-Tag monoclonal antibody	MBL	D291-7	HRP-conjugated
BCA protein assay kit	Thermo Fisher Scientific	23227	Measurement of protein concentration
Brain heart infusion broth	Becton, Dickinson	237500	Bacterial culture medium
CBB R-250	Wako	031-17922	For biofilm staining
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
DNA polymerase	Takara	R045A	High-fidelity DNA polymerase
DNA sequencing	Eurofins Genomics
ECL substrate	Bio-Rad	170-5060	For western blotting
EDTA (0.5 M pH 8.0)	Wako	311-90075	Tris-EDTA buffer preparation
Electroporation cuvette	Bio-Rad	1652086	0.2 cm gap
Electroporator	Bio-Rad	1652100
EtBr solution	Nippon Gene	315-90051	For agarose gel electrophoresis
Gel band cutter	Nippon Genetics	FG-830
Gel extraction kit	Nippon Genetics	FG-91202	DNA extraction from agarose gel
Imager	GE Healthcare	29083461	For SDS-PAGE and western blotting
Imidazole	Wako	095-00015	Binding buffer and elution buffer preparation
Incubator	Nippon Medical & Chemical Instruments	EZ-022	Temperature setting: 4 °C
Incubator	Nippon Medical & Chemical Instruments	LH-100-RDS	Temperature setting: 37 °C
Membrane filter	Merck Millipore	JGWP04700	0.2 µm diameter
Microcentrifuge	Kubota	3740
NaCl	Wako	191-01665	Preparation of binding buffer and elution buffer
NaH2PO4·2H2O	Wako	192-02815	Preparation of binding buffer and elution buffer
NaOH	Wako	198-13765	Preparation of binding buffer and elution buffer
(NH4)2SO4	Wako	015-06737	Ammonium sulfate precipitation
Ni-charged resin	Bio-Rad	1560133	For IMAC
PCR primers	Eurofins Genomics	Custom-ordered
Protein standard	Bio-Rad	161-0381	For SDS-PAGE and western blotting
Solvent filtration apparatus	As One	FH-1G
Spectinomycin	Wako	195-11531	Antibiotics; use at 100 μg/mL
Sterile syringe filter	Merckmillipore	SLGV004SL	0.22 µm diameter
Streptococus mutans ΔgtfC	Stock strain in the lab.		gtfC replaced with spcr
Streptococus mutans UA159	Stock strain in the lab.		S. mutans ATCC 700610, Wild-type strain
Sucrose	Wako	196-00015	For biofilm development
TAE (50 × )	Nippon Gene	313-90035	For agarose gel electrophoresis
Thermal cycler	Bio-Rad	PTC-200
Tris-HCl (1 M, pH 8.0)	Wako	314-90065	Tris-EDTA buffer preparation