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-1 ampicillin. Inoculate each flask with 20 mL of the overnight culture obtained from step 1.1. Incubate them at 37 &#176;C with continuous shaking at 200 rpm until the optical density at 600 nm reaches 0.6.</li>
	<li>Take a 1 mL aliquot from one of the flasks (uninduced control sample), pellet using a benchtop centrifuge (13,000 x&#160;g, 4 &#176;C, 10 min) and discard the supernatant. Store the bacterial pellet at -20 &#176;C for subsequent SDS-PAGE analysis.</li>
	<li>Induce protein expression by adding 1 mM of isopropyl &#946;-D-thiogalactoside (IPTG) to each flask with culture. Continue incubating the flasks at 37 &#176;C in a shaker at 200 rpm for an additional 4 h.</li>
	<li>Harvest the cells by centrifugation at 5,000 x&#160;g for 15 min at 4 &#176;C and discard the supernatant. 
	NOTE: At this point, the procedure can be paused by placing the cell pellet into a -80 &#176;C freezer for storage.</li>
</ol>
2. Isolation and Denaturation of Inclusion Bodies
<ol>
	<li>Transfer all the cell pellets obtained in step 1.5 to a 250 mL beaker and add 100 mL of buffer A (10 mM Tris-HCl, pH 8.0, 150 mM NaCl). Allow the cells to thaw completely (if frozen), and resuspend the pellet. Keep the sample on ice unless indicated otherwise.</li>
	<li>Pass the resuspended cells through a high-pressure homogeniser three times to lyse the cells and ensure complete shearing of the genomic DNA.</li>
	<li>Centrifuge the lysate at 10,000 x&#160;g for 15 min at 4 &#176;C. Collect a 1 mL sample of the supernatant (soluble fraction) and store it at -20 &#176;C for subsequent SDS-PAGE analysis. Discard the rest of the supernatant and place the pellet on ice. 
	NOTE: This pellet is white in color (easily distinguishable from unbroken cells which are the shade of brown in color) and contains inclusion bodies.</li>
	<li>Resuspend the inclusion bodies pellet thoroughly in 20 mL of ice-cold buffer B (10 mM Tris-HCl pH 8.0, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 1% Triton X-100). This facilitates solubilisation of the membrane and membrane proteins. Vortex for 1 - 2 min to aid resuspension.</li>
	<li>Centrifuge the sample at 10,000 x&#160;g for 15 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet thoroughly in 20 mL of ice-cold buffer B by vortexing the tube for 1 - 2 min. Ensure that the pellet is broken into small pieces. Pipet the sample up and down to aid pellet resuspension. 
	NOTE: It is important to resuspend the pellet thoroughly to obtain inclusion bodies free from impurities.</li>
	<li>Repeat step 2.5. If the supernatant is cloudy or coloured, repeat steps 2.6 and 2.5 (in that order) until the supernatant is clear and colourless.</li>
	<li>Resuspend the pellet in 20 mL of ice-cold buffer C (10 mM Tris-HCl pH 8.0, 0.2 mM PMSF) by vortexing the tube for 1 - 2 min.</li>
	<li>Repeat step 2.5.</li>
	<li>Add 25 mL of ice-cold denaturing buffer D (10 mM Tris-HCl pH 8.0, 8 M urea, 10 mM dithiothreitol (DTT), 0.2 mM PMSF) to dissolve the inclusion bodies pellet. Resuspend thoroughly by vortexing for 1 - 2 min, or until the pellet is broken into small pieces.</li>
	<li>Mix the suspension by axial rotation with a rotation rate of 30 rpm for 30 - 120 min at 4 &#176;C.</li>
	<li>Clarify the denatured protein solution by centrifugation at 30,000 x g, 4 &#176;C for 30 min, place the supernatant on ice and discard the pellet.</li>
	<li>Measure the protein concentration using the Bradford assay.24 Take an aliquot for SDS-gel analysis and place it at -20 &#176;C. 
	NOTE: At this point, the procedure can be paused by snap-freezing the aliquoted sample in liquid nitrogen and keep it at -80 &#176;C for storage.</li>
</ol>
3. Protein Refolding
<ol>
	<li>Prepare 250 mL of buffer E (3 M urea, 100 mM Tris-HCl pH 8.0, 0.4 M L-arginine monohydrochloride, 20 mM reduced L-glutathione, 2 mM oxidized L-glutathione) in a 500 mL beaker and cool the buffer down to 4 &#176;C.</li>
	<li>Add 60 mg of denatured protein mix containing His6-Tlp3-LBD obtained in step 2.13 to 250 mL of buffer E, while stirring the buffer at 500 rpm. Incubate the refolding mix at 4 &#176;C with continuous stirring at 500 rpm for 24 - 48 h. The final protein concentration in this step is 0.2 mg mL-1.</li>
	<li>Prepare 7 L of buffer A in an 8 - 10 L bucket and cool it down to 4 &#176;C in preparation for the next step (step 3.4) that will take place in 24 - 48 h (see the flowchart in Figure 1).</li>
	<li>Immerse an ~50 cm piece of dialysis tubing of 28 mm inflated diameter, with molecular weight cut-off at 12 - 14 kDa into 200 mL of 10 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) and heat it over a gas flame until boiling. Rinse the dialysis membrane with 200 mL of ddH2O.</li>
	<li>Clamp one end of the dialysis membrane with a dialysis tubing closure and transfer the 250 mL refolding mixture obtained in step 3.2 into the dialysis tube. Close the open end with another clamp. Check the integrity of the membrane to ensure there are no leaks.</li>
	<li>Place the dialysis tube in a dialysis bucket with the pre-cooled 7 L of buffer A prepared in step 3.3. Place a magnetic stir bar, ensuring that it will not touch the dialysis tubing while stirring.</li>
	<li>Dialyse the sample at 4 &#176;C with continuing stirring at 500 rpm and change the buffer at least four times over a period of 12 h. After the last buffer change, leave the sample to dialyse overnight. 
	NOTE: During dialysis, the excess of urea (~3 M) is gradually removed from the denatured protein solution, which allows the protein to refold. Heavy protein precipitation can be observed at the end of dialysis.</li>
	<li>Remove the dialysis tube from the bucket and transfer its contents into a 500 mL beaker. Keep the protein solution on ice, unless indicated otherwise.</li>
	<li>Filter the protein solution through a 0.43 &#181;m pore size membrane into a 500 mL glass bottle to remove any precipitated protein.</li>
</ol>
4. Purification of His6-tagged Protein Using Immobilised Metal Ion Affinity Chromatography
<ol>
	<li>Charge and equilibrate a 5 mL prepacked chelating column.
	<ol>
		<li>Connect the column to a peristaltic pump, ensuring that there is no air trapped in the connecting tubing.</li>
		<li>Wash the column by passing through 25 - 50 mL of ddH2O.</li>
		<li>Charge the column by loading 3 mL of 0.1 M NiCl2 and then passing through 25 mL of ddH2O.</li>
		<li>Equilibrate the column with 25 mL of buffer F (loading buffer, 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole).</li>
	</ol>
	</li>
	<li>Adjust the refolded protein sample obtained in step 3.9 to the composition of buffer F by adding 2.5 mL of 1 M Tris-HCl pH 8.0, 25 mL of 5 M NaCl and 2.5 mL of 2 M imidazole stock solutions.</li>
	<li>Load the sample onto the column at a rate of 5 mL/min or less and discard the flow-through (unbound proteins).</li>
	<li>Wash the column with 50 - 100 mL of buffer F to remove non-specifically bound proteins and discard the flow-through.</li>
	<li>Elute His6-Tlp3-LBD with 25 mL of buffer G (elution buffer, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole) and pool the flow-through fractions containing the protein (as determined by using the Bradford reagent).</li>
	<li>Measure the protein concentration in the pooled sample using the Bradford assay. Take a small aliquot for SDS-PAGE analysis and place at -20 &#176;C.</li>
</ol>
5. His6-tag Removal Using TEV Protease (Optional)
<ol>
	<li>Prepare, and cool down to 4 &#176;C, 4 L of buffer H (TEV cleavage buffer, 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % (v/v) glycerol, 2 mM DTT).</li>
	<li>Cut approximately 5 - 7 cm of dialysis tubing (diameter 2 - 3 cm) and immerse it in 200 mL of ddH2O.</li>
	<li>Add His6-tagged TEV protease to the His6-tag protein prepared in step 4.5 to a final molar ratio 1 TEV: 8 protein.</li>
	<li>Clamp one end of the tubing with a dialysis tubing closure and fill it with the protein/TEV protease mixture. Close the other end and ensure there are no leaks.</li>
	<li>Place the dialysis tube into the pre-cooled 4 L of buffer H (from step 5.1) and incubate it at 4 &#176;C with continuous stirring for 2 h. Change the buffer and continue dialysis overnight to allow the TEV-mediated cleavage reaction to complete.</li>
	<li>In the meantime, prepare and cool (4 &#176;C) 4 L of buffer I (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % (v/v) glycerol) in preparation for the next day.</li>
	<li>After the overnight dialysis, exchange the buffer to buffer I prepared in step 5.6 and dialyse for a further 1 - 2 h at 4 &#176;C.</li>
	<li>Remove the tube from the dialysis bucket, unclip one end of the tube and transfer the protein solution to a 50 mL Falcon tube. Filter the solution through a 0.43 &#181;m pore size syringe filter and keep it on ice, unless stated otherwise.</li>
	<li>Measure the volume of the sample and adjust the Tris, NaCl and imidazole concentration to the composition of buffer F (e.g. for 25 mL of protein solution in buffer J add 0.25 mL of 1 M Tris-HCl pH 8.0, 1.75 mL of 5 M NaCl, and 0.25 mL of 2 M imidazole).</li>
	<li>Prepare a 5 mL HiTrap Chelating HP column as described in step 4.1.</li>
	<li>Load the sample onto the column (flow rate: 5 mL/min) and collect the flow-through, which contains the untagged Tlp3-LBD (residues 42 - 291). Uncleaved protein, the cleaved His6-tag and His6-TEV are retained by the column.</li>
	<li>Wash the column with 5 mL buffer F (loading buffer) to allow all untagged protein to flow through and collect the eluate.</li>
	<li>Pool the eluate obtained in steps 5.11 and 5.12 and measure the protein concentration. Take a small aliquot and store it at -20 &#176;C for SDS-PAGE analysis.</li>
</ol>
6. Size-exclusion Chromatography (Gel Filtration) of Tlp3-LBD
<ol>
	<li>Prepare 1 L of buffer A (10 mM Tris-HCl pH 8.0, 150 mM NaCl) and filter it using a 0.43 &#181;m membrane.</li>
	<li>Equilibrate a 26/60 size-exclusion column with buffer A at a flow rate of 4 mL/min.</li>
	<li>Concentrate the protein sample obtained in step 5.13 to a volume of 3 - 4 mL using 15 mL centrifugal concentrator with a 10 kDa cut-off (4 &#176;C, 4,000 x&#160;g).</li>
	<li>Clarify the protein solution by centrifugation at 13,000 x&#160;g, 4 &#176;C for 30 min to remove any precipitated protein.</li>
	<li>Load the sample onto the pre-equilibrated 26/60 gel filtration column. Perform chromatography in buffer A at a flow rate of 4 mL/min. Monitor the UV trace. Tlp3-LBD elutes at a retention volume of 210 - 220 mL. Pool the peak fractions.</li>
	<li>Measure the protein concentration. Take a small aliquot for SDS-PAGE analysis.</li>
</ol>
7. SDS-PAGE Analysis of Samples Collected at Various Stages of Protein Purification
<ol>
	<li>Prepare 1 L of SDS-PAGE running buffer (buffer J), containing 25 mM Tris, 250 mM glycine pH 8.3, and 0.1 % (w/v) sodium dodecyl sulfate (SDS).</li>
	<li>Prepare 10 mL of 5x SDS-PAGE loading buffer (buffer K), containing 0.25 M Tris-HCl pH 6.8, 10% (w/v) SDS, 50% (v/v) glycerol, 1 mM DTT, and 0.05% bromophenol blue.</li>
	<li>Allow the aliquots collected during the different steps of the purification (steps 1.3, 2.3, 2.13, 4.6, 5.13, 6.6) to thaw.</li>
	<li>For each sample, mix the volume corresponding to 15 &#181;g of total protein with 2 &#181;L of 5x&#160;SDS-PAGE loading buffer, and adjust the volume to 10 &#181;L with ddH2O.</li>
	<li>Heat the samples at 95 &#176;C for 5 - 10 min, spin them at 10,000 x&#160;g for 30 s and transfer the samples into a clean tube.</li>
	<li>Prepare an SDS-PAGE gel, using 15% polyacrylamide separating gel (for 5 mL: 2.5 mL 30% (w/v) bis-acrylamide, 1.2 mL 1.5 M Tris-HCl pH 8.8, 1.2 mL ddH2O, 50 &#181;L 10% (w/v) SDS, 50 &#181;L 20% (w/v) ammonium persulfate (APS), 3 &#181;L tetramethylethylenediamine (TEMED) and 5% polyacrylamide stacking gel (for 2 mL: 340 &#181;L 30% (w/v) bis-acrylamide, 250 &#181;L 1 M Tris pH 6.8, 1.37 mL ddH2O, 20 &#181;L 10% (w/v) SDS, 20 &#181;L 20% (w/v) APS, 2 &#181;L TEMED).</li>
	<li>Assemble the electrophoresis apparatus and fill it with 1x SDS page running buffer as per manufacturer&#39;s recommendation.</li>
	<li>Load a protein molecular weight marker and the samples prepared in step 7.5. Perform the electrophoresis at a constant current of 25 mA.</li>
	<li>Transfer the gel into a container and rinse it with ddH2O. Add 150 mL of the Coomassie Blue staining solution containing 25% (v/v) isopropanol, 10% (v/v) acetic acid and 0.03% (w/v) Brilliant Blue R-250. Place the container on a horizontal rotation platform for 30 min at room temperature.</li>
	<li>Rinse the gel with ddH2O and place into the destaining solution containing 5% (v/v) methanol and 7% (v/v) acetic acid. Destain while mixing using a horizontal rotation platform for 1 h.</li>
	<li>Image the gel and analyse.</li>
</ol>
8. Circular Dichroism (CD) Spectroscopy Analysis of Secondary Structure of refolded Pure Protein
<ol>
	<li>Transfer 0.5 - 1 mg of refolded protein obtained in step 6.6 into a dialysis tube and place it in a dialysis bucket containing 5 L of buffer L (50 mM sodium phosphate pH 7.4), precooled to 4 &#176;C. Dialyse the sample at 4 &#176;C with continuous stirring and perform at least 3 - 4 buffer changes over 2 - 4&#160;h before leaving the sample to dialyse overnight.</li>
	<li>Concentrate the dialysed protein solution to 0.06 mg mL-1 using a 10 kDa cut-off centrifugal concentrator.</li>
	<li>Clarify the solution by centrifugation at 13,000 x&#160;g, 4 &#176;C for 30 min.</li>
	<li>Record the far-UV CD spectrum of the sample at 25 &#176;C over a wavelength range of 200 - 260 nm using a spectropolarimeter with a scan rate of 20 nm min-1. Use the dialysis buffer as a blank control. Repeat recording in triplicate and generate the averaged spectrum.</li>
	<li>Calculate the secondary structure content by deconvoluting the CD spectra using the CDSSTR program from the DichroWeb sever25 (http://dichroweb.cryst.bbk.ac.uk).</li>
</ol>

The authors declare no competing financial interests.

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>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>MERK MILLIPORE</td>
			<td>1064041000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>G-BIOSCIENCES</td>
			<td>A051-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)&#160;</td>
			<td>MERCK</td>
			<td>52332</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>AMRESCO</td>
			<td>694</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>AST0487</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>AMRESCO</td>
			<td>VWRC0568</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>C-1029</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginine monohydrochloride</td>
			<td>SIGMA-ALDRICH</td>
			<td>A5131</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced L-glutathione</td>
			<td>SIGMA-ALDRICH</td>
			<td>G4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxidized L-glutathione</td>
			<td>SIGMA-ALDRICH</td>
			<td>G4376</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>ASTRAL SCIENTIFIC PTY LTD</td>
			<td>BIOGB0232</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel chloride (NiCl2)</td>
			<td>SIGMA-ALDRICH</td>
			<td>339350</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>AMRESCO</td>
			<td>VWRC0167</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L4509</td>
			<td></td>
		</tr>
		<tr>
			<td>Unstained Protein Ladder, Broad Range (10-250 kDa)</td>
			<td>NEW ENGLAND BIOLABS</td>
			<td>P7703</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultracel centrifugal concentrator (Millipore)</td>
			<td>MERCK</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<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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Biochemistry

17.7K Views.  Monash Biomedicine Discovery Institute. 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.

Video: Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.