Name: method for efficient refolding and purification of chemoreceptor ligand binding domain
Uploaded: 2017-12-12T15:00:00
Description: Watch this Scientific Journal Video about Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain at JoVE.com

We thank Yu C. Liu for his early work on the Tlp3-LBD production. Mayra A. Machuca is indebted to Departamento Admistrativo de Ciencia, Tecnolog&#237;a e Innovaci&#243;n COLCIENCIAS for a doctoral scholarship.

1. Expression of His6-Tlp3-LBD&#160;in E. coli
<ol>
	<li>Inoculate 150 mL of sterile Luria-Bertani (LB) broth containing 50 &#181;g mL-1 ampicillin with BL21-Codon-Plus(DE3)-RIPL cells transformed with the pET151/D-TOPO vector for expression of His6-Tlp3-LBD (amino acid residues 42-291), and incubate it at 37 &#176;C in a shaker (orbit diameter, 25 mm) with 200 rpm overnight.</li>
	<li>Prepare six 2 L Erlenmeyer flasks containing 800 mL of sterile LB broth and 50 &#181;g mL<sup...

A simple procedure for expression and refolding from inclusion bodies of the periplasmic LBD of the bacterial chemoreceptor Tlp3 is presented. Preparation of the pure protein involves over-expression of the pET-plasmid-encoded gene in E. coli, purification and solubilisation of inclusion bodies, refolding of the denatured protein and its purification by the consecutive affinity and size-exclusion chromatography steps. The urea-facilitated denaturation and dilution/dialysis-mediated refolding are the critical ste...

Chemotaxis and motility have been shown to play important roles in Campylobacter jejuni pathogenesis by promoting bacterial colonisation and invasion of the host1,2,3. Chemotaxis allows bacteria to move towards an optimal environment for growth, as guided by chemical signals. This process involves recognition of intracellular and environmental chemical cues by a set of proteins termed chemoreceptors, or transducer-like proteins (Tlps). Most chemoreceptors are membrane-embedded proteins with an extracytoplasmic ligand binding domain (LBD), a transmembrane domain and a cytoplasmic signalling domain, the latter of which interacts with the cytosolic signalling proteins that transmit the signal to the flagellar motors4,5,6,7.
Eleven different chemoreceptors have been identified in the C. jejuni genome4,8. To date, only some of these chemoreceptors have been characterised and the ligand specificity of Tlp19, Tlp310,11, Tlp411, Tlp712, and Tlp1113 is known. Identification of natural ligands of the remaining chemoreceptors in this species, and numerous chemoreceptors in other bacteria, can be greatly facilitated by the production of folded and highly pure recombinant chemoreceptor LBDs14,15,16. However, attempts to heterologously express periplasmic LBDs of bacterial chemoreceptors in Escherichia coli often result in their targeting into inclusion bodies17,18,19. Nevertheless, this phenomenon can facilitate easy isolation and recovery of the protein in hand. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic LBD of the C. jejuni chemoreceptor Tlp3 as an example. This example was chosen because Tlp3-LBD belongs to the dCACHE family20 of sensing domains which are abundantly found in two-component histidine kinases and chemoreceptors in prokaryotes20,21,22,23.
In this approach, the expression construct, based on a pET151/D-TOPO vector, has been designed to incorporate an N-terminal His6-tag followed by a tobacco etch virus (TEV) protease cleavage site, for subsequent tag removal19. The protocol describes protein overexpression in E. coli, isolation and urea-mediated solubilisation of inclusion bodies, and protein refolding by urea depletion. Following refolding, the sample is purified by affinity chromatography, with optional tag removal and size-exclusion chromatography. The folded state of the purified protein is confirmed using circular dichroism spectroscopy. This is a modified version of the method that has been previously developed to recover and purify the LBD of a different chemoreceptor, Helicobacter pylori TlpC19. This procedure, summarised in Figure 1, yields 10 - 20 mg of pure, untagged Tlp3-LBD per 1 L of bacterial culture, with a protein purity of &#62;90% as estimated by SDS-PAGE.

<table>
	<tbody>
		<tr>
			<td>Tris base</td>
			<td>AMRESCO</td>
			<td>497</td>
			<td></td>
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			<td>Sodium chloride (NaCl)</td>
			<td>MERK MILLIPORE</td>
			<td>1064041000</td>
			<td></td>
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		<tr>
			<td>Ampicillin</td>
			<td>G-BIOSCIENCES</td>
			<td>A051-B</td>
			<td></td>
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			<td>Phenylmethanesulfonyl fluoride (PMSF)&#160;</td>
			<td>MERCK</td>
			<td>52332</td>
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			<td>Triton x-100</td>
			<td>AMRESCO</td>
			<td>694</td>
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			<td>Isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>AST0487</td>
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			<td>Urea</td>
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			<td>VWRC0568</td>
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			<td>Dithiothreitol</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>C-1029</td>
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			<td>L-arginine monohydrochloride</td>
			<td>SIGMA-ALDRICH</td>
			<td>A5131</td>
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		<tr>
			<td>Reduced L-glutathione</td>
			<td>SIGMA-ALDRICH</td>
			<td>G4251</td>
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		<tr>
			<td>Oxidized L-glutathione</td>
			<td>SIGMA-ALDRICH</td>
			<td>G4376</td>
			<td></td>
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		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td>&#160;7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td>10049-21-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)</td>
			<td>AMRESCO</td>
			<td>VWRC20302.260</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>SIGMA-ALDRICH</td>
			<td>I2399</td>
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			<td>Glycerol</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>BIOGB0232</td>
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			<td>Nickel chloride (NiCl2)</td>
			<td>SIGMA-ALDRICH</td>
			<td>339350</td>
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			<td>Glycine</td>
			<td>AMRESCO</td>
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			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L4509</td>
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			<td>Unstained Protein Ladder, Broad Range (10-250 kDa)</td>
			<td>NEW ENGLAND BIOLABS</td>
			<td>P7703</td>
			<td></td>
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			<td>Amicon Ultracel centrifugal concentrator (Millipore)</td>
			<td>MERCK</td>
			<td>UFC901096</td>
			<td></td>
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		<tr>
			<td>50 mL Falcon tube</td>
			<td>FALCON</td>
			<td>BDAA352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>LIVINGSTONE INTERNATIONAL PTY</td>
			<td>Dialysis</td>
			<td></td>
		</tr>
		<tr>
			<td>Snakeskin dialysis tubing</td>
			<td>THERMO SCIENTIFIC&#8482;</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepacked HiTrap Chelating HP column</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>17-0408-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EmulsiFlex-C5 high-pressure homogeniser</td>
			<td>AVESTIN</td>
			<td>EmulsiFlex&#8482; - C5</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump P-1</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>18-1110-91&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 HiLoad 26/60 size-exclusion column&#160;</td>
			<td>GE HEALTHCARE LIFE SCIENCES</td>
			<td>28989336</td>
			<td></td>
		</tr>
		<tr>
			<td>JASCO J-815 spectropolarimeter</td>
			<td>JASCO</td>
			<td>J-815</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for efficient refolding and purification of chemoreceptor ligand binding domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

Recombinant expression of His6-Tlp3-LBD in E. coli resulted in protein deposition in inclusion bodies. The expression yield from 1 L of bacterial culture calculated in step 2.13 was approximately 100 mg of His6-Tlp3-LBD deposited in inclusion bodies. The protein isolation procedure, described here and illustrated in Figure 1, consists of the solubilisation of inclusion bodies, protein refolding and purification, by means of affi...

Watch this Scientific Journal Video about Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain at JoVE.com

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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