This work was supported by the Japan Society for the Promotion of Science (JSPS) (grant numbers 16K15860 and 19K10471 to T. M., 17K12032 to M. I., and 18K09926 to N. H.) and the SECOM Science and Technology Foundation (SECOM) (grant number 2018.09.10 No. 1).

NOTE: Generation of S. mutans &#8710;gtfC, in which the entire coding region of the gtfC gene is replaced with spcr, must be completed prior to performing these protocols. Refer to the published article for details on generation5.
1. Primer design
<ol>
	<li>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 schematically illustrated in Figure 2.
	<ol>
		<li>Design primers (gtfC-reverse and spcr-forward) for the attachment of His-tag-coding sequence, intervening between the gtfC gene and spcr (Figure 2A).
		<ol>
			<li>Include GS linker and His-tag-coding sequence into both gtfC-reverse and spcr-forward primers, in which 24 bases at the 5&#8217; regions are complementary to each other. 
			NOTE: The amino acid sequence of the GS linker and His-tag (Gly-Gly-Gly-Gly-Ser-His-His-His-His-His-His) is attached to the C-terminus of GTF-SI through gene translation.</li>
			<li>Design a gtfC-forward primer to target the gtfC gene in the S. mutans WT genome &#62;1 kb downstream of the gene and the spcr-reverse primer to target the downstream flanking region of spcr in the S. mutans &#8710;gtfC. Set the melting temperature8 (Tm) of the gtfC-forward and spcr-reverse primers to match with the melting temperatures of the gtfC-reverse and spcr-forward primers, respectively.</li>
		</ol>
		</li>
		<li>Design nested primers (nested-forward and nested-reverse) for the second PCR (Figure 2B).</li>
		<li>Design primers (gtfC-forward and colony-reverse) specific for the genomic DNA of the transformant (Figure 2C; the primers are used for colony PCR). 
		NOTE: The amplicons straddle the border between gtfC and spcr. The gtfC-forward primer can be applied as the forward primer.</li>
		<li>Design primers (up-forward and down-reverse) for final confirmation of the generation of S. mutans His-gtfC (Figure 2C; the PCR product amplified by the primers is used for DNA sequencing).</li>
	</ol>
	</li>
</ol>
2. Genomic DNA Extraction from S. mutans
NOTE: Each S. mutans strain should be cultured in brain heart infusion (BHI) medium at 37 &#176;C under anaerobic conditions. The mutant strains of S. mutans &#8710;gtfC and S. mutans His-gtfC are cultured in BHI supplemented with 100 &#181;g/mL spectinomycin.
<ol>
	<li>Streak S. mutans WT and S. mutans &#8710;gtfC separately onto each of the BHI agar plates. Incubate them overnight at 37 &#176;C.</li>
	<li>Pick single colonies of S. mutans WT or S. mutans &#8710;gtfC using a sterilized toothpick and inoculate into 1.5 mL of BHI broth. Incubate them overnight at 37 &#176;C. 
	NOTE: The colonies may be used as a source of template DNA for the first PCR (step 3.1).</li>
	<li>Transfer 1 mL of each bacterial culture to a 1.5 mL microcentrifuge tube. Centrifuge the culture for 1 min at 15,000 x g.</li>
	<li>Remove the supernatant and add 1 mL of the Tris-EDTA buffer (pH 8.0) to resuspend the cell pellet.</li>
	<li>Centrifuge the suspension for 1 min at 15,000 x g. Remove the supernatant. Resuspend the cell pellet in 50 &#181;L of Tris-EDTA buffer (pH 8.0).</li>
	<li>Heat the suspension using a block incubator for 5 min at 95 &#176;C. Centrifuge the suspension for 5 min at 15,000 x g.</li>
	<li>Transfer the supernatant to a new 1.5 mL microcentrifuge tube (the supernatant will serve as template DNA for the first PCR).</li>
</ol>
3. PCR Amplification
NOTE: Table 1, Table 2, and Table 3 summarize the PCR primers, reagents, and amplification cycles, respectively.
<ol>
	<li>Perform the first PCR using the S. mutans WT and S. mutans &#8710;gtfC genome as the PCR templates. Amplify the regions harboring the downstream part of the gtfC gene and those harboring spcr using the primers described in step 1.1.1.2 (Figure 2A).</li>
	<li>Electrophorese each PCR product on 1% agarose gel. Excise the desired DNA fragments of approximately 1,000 bp and 2,000 bp. Purify the fragments from the gels using silica membrane-based gel extraction method. 
	NOTE: The basic procedures for PCR9, DNA gel electrophoresis10, and DNA purification11 are detailed elsewhere.</li>
	<li>Perform a second PCR using the products of the first PCR (approximately equimolar) as PCR templates with the nested-forward and nested-reverse primers (Figure 2B).</li>
	<li>Electrophorese one-tenth of the PCR mixture on the agarose gel. Confirm the generation of the appropriate amplicon of approximately 3,000 bp.</li>
	<li>Precipitate the remaining PCR product. 
	NOTE: Purification of the PCR product is not necessary, because non-specific amplicons generated by the second PCR do not interfere with homologous recombination.
	<ol>
		<li>Add nuclease-free water to the PCR mixture and bring the total volume to 100 &#181;L, followed by 0.1 volume (10 &#181;L) of 3 M sodium acetate (pH 5.2) and 2.5 volumes (250 &#181;L) of absolute ethanol. Mix and store the mixture for 10 min at room temperature (RT).</li>
		<li>Centrifuge the sample for 20 min at 15,000 x g at 4 &#176;C. Discard the supernatant. Add 1 mL of 70% ethanol to wash the DNA pellet.</li>
		<li>Centrifuge the sample for 5 min at 15,000 x g at 4 &#176;C. Discard the supernatant. Air-dry and dissolve the DNA pellet with 10 &#181;L of nuclease-free water.</li>
	</ol>
	</li>
</ol>
4. Cell Transformation
<ol>
	<li>Prepare competent S. mutans WT to introduce the second PCR product by following the steps described in the previous publication5. Store the cells at -80 &#176;C. 
	NOTE: For this experiment, the competent S. mutans WT cells have already been preserved in -80 &#176;C to generate S. mutans &#8710;gtfC.</li>
	<li>Mix 5 &#956;L of the concentrated second PCR product to the 50 &#956;L aliquot of ice-cold competent cells frozen previously. Add the mixture into electroporation cuvettes. Give a single electric pulse (1.8 kV, 2.5 ms, 600 &#937;, 10 &#181;F) to the cells using the electroporation apparatus.</li>
	<li>Resuspend the cells in 500 &#956;L of BHI broth. Immediately, spread 10&#8211;100 &#956;L of the suspension onto BHI agar plates containing spectinomycin. Incubate the plates for 2&#8211;6 days at 37 &#176;C until colonies have grown sufficiently to be picked up.</li>
</ol>
5. Verification of Genome Recombination and Storage
<ol>
	<li>Perform colony PCR, using gtfC-forward and colony-reverse primers to screen for the recombination. Electrophorese each colony PCR product on an agarose gel. Confirm the DNA fragment of approximately 1,500 bp.</li>
	<li>Pick one of the positive colonies using a sterilized toothpick and subculture the cells overnight in 2 mL of BHI broth containing spectinomycin.</li>
	<li>Mix 0.8 mL of bacterial culture with 0.8 mL of sterile 50% glycerol and stock at -80 &#176;C or -20 &#176;C.</li>
	<li>Transfer the remaining 1 mL of cell suspension to a 1.5 mL microcentrifuge tube. Repeat steps 2.3&#8211;2.7 to extract the genomic DNA from the cells.</li>
	<li>Perform PCR using the up-forward and down-reverse primers and genomic DNA as a template. Repeat step 3.2 to purify the amplified DNA product from the gels.</li>
	<li>Determine the DNA sequence of the purified amplicon by DNA sequencing. 
	NOTE: Make sure to confirm the generation of S. mutans His-gtfC by DNA sequencing.</li>
</ol>
6. Purification of Polyhistidine-tagged GTF-SI
<ol>
	<li>Streak S. mutans His-gtfC onto a spectinomycin-containing BHI agar plate. Incubate them overnight at 37 &#176;C.</li>
	<li>Pick up a single colony using a sterilized toothpick and subculture cells in 3 mL of BHI broth. Incubate overnight at 37 &#176;C.</li>
	<li>Prepare two conical flasks with 1 L of BHI broth without spectinomycin. Inoculate 1 mL of the overnight culture suspension into 1 L of the BHI broth. Incubate overnight at 37 &#176;C.</li>
	<li>Concentrate proteins from the culture supernatant by ammonium sulfate precipitation. 
	NOTE: Extract from cell bodies if a target protein is intracellular. The basic procedures for ammonium sulfate precipitation are detailed elsewhere12.
	<ol>
		<li>Centrifuge the bacterial culture suspension for 20 min at 10,000 x g at 4 &#176;C. Recover the culture supernatant into a 3 L glass beaker.</li>
		<li>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 &#176;C with stirring. 
		NOTE: The precipitate formation can be continued overnight.</li>
		<li>Centrifuge the ammonium sulfate-precipitated solution at 15,000 x g for 20 min at 4 &#176;C. Decant the supernatant.</li>
		<li>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).</li>
		<li>Dialyze the suspension against 2,500 mL of the binding buffer using a regenerated cellulose dialysis tubing, at 4 &#176;C, with stirring. Replace the dialysis solution after 2 h and continue dialysis overnight.</li>
		<li>Replace the dialysis solution again. Continue to dialyze for an additional 2 h.</li>
	</ol>
	</li>
	<li>Centrifuge the dialyzed suspension at 20,000 x g for 10 min at 4 &#176;C. Filter the supernatant through a membrane filter using suction filtration equipment. Transfer the filtrate to a 75 cm2 flask.</li>
	<li>Fractionate the polyhistidine-tagged GTF-SI from the filtrated suspension by immobilized metal affinity chromatography (IMAC).
	<ol>
		<li>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.</li>
		<li>Add 3 mL of distilled water into the column to wash the resin. Add 5 mL of the binding buffer to equilibrate the resin.</li>
		<li>Shut off the flow with a Hoffmann pinch cock. Add 5 mL of the filtered suspension from step 6.5 to make the slurry.</li>
		<li>Add all the slurry to the remaining filtered suspension. Swirl the mixture gently for 30 min at 4 &#176;C.</li>
		<li>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.</li>
		<li>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 &#176;C. IMAC resin can be reused. Refer to the instructions for the IMAC resin.</li>
	</ol>
	</li>
	<li>Replace the elution buffer with the storage buffer (50 mM phosphate buffer, pH 6.5) and concentrate the recombinant GTF-SI solution to approximately 1 mL using a centrifugal ultrafiltration unit. Store the recombinant GTF-SI solution at 4 &#176;C. 
	NOTE: Confirm the polyhistidine-tagged GTF-SI purification using SDS-PAGE13 and western blotting14 by employing a horseradish peroxidase-conjugated anti-polyhistidine monoclonal antibody. Addition of CHAPS (final concentration, 0.1%) to the eluent may be required to avoid nonspecific adsorption to the unit, depending on the target protein.</li>
</ol>
7. Functional Restoration by Recombinant GTF-SI
<ol>
	<li>Streak each S. mutans strain onto a BHI agar plate individually. Incubate the plates overnight at 37 &#176;C.</li>
	<li>Using a sterilized toothpick, pick up a single colony to inoculate into 2 mL of BHI broth. Incubate overnight at 37 &#176;C.</li>
	<li>Use 20 &#956;L of the overnight culture of S. mutans &#8710;gtfC to inoculate 2 mL of BHI broth containing 1% sucrose without antibiotics in a glass test tube. Add the GTF-SI or an equivalent volume of the vehicle to the S. mutans &#8710;gtfC culture, and culture the cells with the tube placed in an inclined position overnight. 
	NOTE: Sterilize the GTF-SI solution with a sterile syringe filter and determine protein concentration using a bicinchoninic acid protein assay15 before use.</li>
	<li>Agitate the culture suspensions with a vortex mixer for 10 s. Decant the suspensions. Wash the test tubes with a sufficient amount of distilled water.</li>
	<li>Stain the biofilms on the tube wall with 1 mL of 0.25% Coomassie brilliant blue (CBB).</li>
	<li>Decant the staining solution after 1 min. Wash the test tubes with a sufficient amount of distilled water. Air-dry the test tubes.</li>
</ol>

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The authors have nothing to disclose.

The design of primers is the most critical step in the protocol. The sequences of the gtfC-reverse and spcr-forward primers were automatically determined based on the sequences of both the 3&#8242; end region of gtfC and the 5&#8242; end region of spcr. Each primer includes 24 complementary bases that encode a GS linker and a His-tag-coding sequence at their 5&#39; regions. Disruption of the native regulatory sequences located in the upstream flanking regions can ...

Analysis of a gene&#39;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 protein is usually isolated from the cells that natively synthesizes the protein through a complex series of steps, which may lead to loss of the gene product. To overcome these issues, a simple procedure has been developed for gene product purification following a gene disruption method2, PCR-based DNA splicing method3&#160;(designated two-step fusion PCR), and electroporation for genetic transformation in Streptococcus mutans. Addition of a polyhistidine tag (His-tag) to the C-terminus of the gene product facilitates its purification by immobilized metal affinity chromatography (IMAC).
To isolate the His-tag-expressing strain, the entire genomic DNA of the gene of interest (in this His-tag-expressing gene-disrupted strain) is replaced with an antibiotic-resistant marker gene. The procedure for generating the His-tag-expressing strainis nearly identical to that for generating a gene-disrupted strain as described previously4,5. Therefore, the methods for gene disruption and gene product isolation should be performed as serial experiments for the functional analysis.
In the present work, a polyhistidine-coding sequence is attached to the 3&#8242; end of the gtfC (GenBank locus tag SMU_1005) gene, encoding glucosyltransferase-SI (GTF-SI) in S. mutans6. Then, expression studies in a streptococcal species were performed. Achieving heterologous gtfC expression by E. coli is difficult, likely because of the high molecular mass of GTF-SI. This strain is named S. mutans His-gtfC. A schematic illustration depicting the organization of the gtfC and spectinomycin resistance gene cassette (spcr)7 loci in wild-type S. mutans (S. mutans WT) and its derivatives is shown in Figure 1. The GTF-SI is a secretory protein that contributes to the development of cariogenic dental biofilm6. Under the presence of sucrose, an adherent biofilm is observed on a smooth glass surface in WT S. mutans strain but not in the S. mutans gtfC-disrupted strain (S. mutans &#916;gtfC)2,5. Biofilm formation is restored in S. mutans &#916;gtfC upon exogenous addition of the recombinant GTF-SI. The strain, S. mutans His-gtfC, is then used to produce the recombinant GTF-SI.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Nippon Genetics</td>
			<td>NE-AG02</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Anaeropack</td>
			<td>Mitsubishi Gas Chemical</td>
			<td>A-03</td>
			<td>Anaerobic culture system</td>
		</tr>
		<tr>
			<td>Anti-His-Tag monoclonal antibody</td>
			<td>MBL</td>
			<td>D291-7</td>
			<td>HRP-conjugated</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td>Measurement of protein concentration</td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>Becton, Dickinson</td>
			<td>237500</td>
			<td>Bacterial culture medium</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>For biofilm staining</td>
		</tr>
		<tr>
			<td>Centrifugal ultrafiltration unit</td>
			<td>Sartorius</td>
			<td>VS2032</td>
			<td>Buffer replacement and protein concentration</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Kubota</td>
			<td>7780II</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>Bio-Rad</td>
			<td>7321010</td>
			<td>For IMAC</td>
		</tr>
		<tr>
			<td>Dialysis membrane clamp</td>
			<td>Fisher brand</td>
			<td>21-153-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>As One</td>
			<td>2-316-06</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Takara</td>
			<td>R045A</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr>
			<td>DNA sequencing</td>
			<td>Eurofins Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECL substrate</td>
			<td>Bio-Rad</td>
			<td>170-5060</td>
			<td>For western blotting</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M pH 8.0)</td>
			<td>Wako</td>
			<td>311-90075</td>
			<td>Tris-EDTA buffer preparation</td>
		</tr>
		<tr>
			<td>Electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>1652086</td>
			<td>0.2 cm gap</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>EtBr solution</td>
			<td>Nippon Gene</td>
			<td>315-90051</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Gel band cutter</td>
			<td>Nippon Genetics</td>
			<td>FG-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel extraction kit</td>
			<td>Nippon Genetics</td>
			<td>FG-91202</td>
			<td>DNA extraction from agarose gel</td>
		</tr>
		<tr>
			<td>Imager</td>
			<td>GE Healthcare</td>
			<td>29083461</td>
			<td>For SDS-PAGE and western blotting</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Wako</td>
			<td>095-00015</td>
			<td>Binding buffer and elution buffer preparation</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>EZ-022</td>
			<td>Temperature setting: 4 &#176;C</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>LH-100-RDS</td>
			<td>Temperature setting: 37 &#176;C</td>
		</tr>
		<tr>
			<td>Membrane filter</td>
			<td>Merck Millipore</td>
			<td>JGWP04700</td>
			<td>0.2 &#181;m diameter</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Kubota</td>
			<td>3740</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Wako</td>
			<td>192-02815</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Wako</td>
			<td>198-13765</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Wako</td>
			<td>015-06737</td>
			<td>Ammonium sulfate precipitation</td>
		</tr>
		<tr>
			<td>Ni-charged resin</td>
			<td>Bio-Rad</td>
			<td>1560133</td>
			<td>For IMAC</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein standard</td>
			<td>Bio-Rad</td>
			<td>161-0381</td>
			<td>For SDS-PAGE and western blotting</td>
		</tr>
		<tr>
			<td>Solvent filtration apparatus</td>
			<td>As One</td>
			<td>FH-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Wako</td>
			<td>195-11531</td>
			<td>Antibiotics; use at 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterile syringe filter</td>
			<td>Merckmillipore</td>
			<td>SLGV004SL</td>
			<td>0.22 &#181;m diameter</td>
		</tr>
		<tr>
			<td>Streptococus mutans &#916;gtfC</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>gtfC replaced with spcr</td>
		</tr>
		<tr>
			<td>Streptococus mutans UA159</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>S. mutans ATCC 700610, Wild-type strain</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td>For biofilm development</td>
		</tr>
		<tr>
			<td>TAE (50 &#215; )</td>
			<td>Nippon Gene</td>
			<td>313-90035</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (1 M, pH 8.0)</td>
			<td>Wako</td>
			<td>314-90065</td>
			<td>Tris-EDTA buffer preparation</td>
		</tr>
	</tbody>
</table>

purification of a high molecular mass protein in streptococcus mutans

Elucidation of a gene&#39;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&#8242; 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.

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...

Watch this Scientific Journal Video about Purification of a High Molecular Mass Protein in Streptococcus mutans at JoVE.com

Purification of a High Molecular Mass Protein in Streptococcus mutans

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.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

purification-of-a-high-molecular-mass-protein-in-streptococcus-mutans

Research

JoVE Journal

Immunology and Infection

5.8K Views.  Tsurumi University School of Dental Medicine. 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.

Video: Purification of a High Molecular Mass Protein in Streptococcus mutans

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...