This research was supported by startup funds at Davidson College.

1. Preparation of media and important buffers
<ol>
	<li>Prepare 500 mL of 40% galactose by adding 200 g of galactose and de-ionized water to a total volume of 500 mL. Use a hot plate or microwave to increase the rate of galactose getting into solution. Sterile filter with a vacuum filtration apparatus consisting of a 0.2 &#181;m filter top attached to a sterile glass bottle. 
	NOTE: All media solutions are prepared using de-ionized water.</li>
	<li>Prepare 500 mL of 40% glucose by adding 200 g of glucose and water to a total volume of 500 mL. Autoclave to sterilize.</li>
	<li>Into each of the four 2 L flasks, add 0.4 g of complete supplement mixture without histidine (CSM-His), 3.35 g yeast nitrogen base with ammonium sulfate (YNB + nitrogen), and 475 mL water. Autoclave. Add 25 mL of 40% glucose to achieve a final glucose concentration of 2%.</li>
	<li>Add, to a glass bottle, 50 g peptone and 25 g yeast extract and water to 375 mL. Autoclave. Add 125 mL of 40% galactose to give 5x yeast peptone (YP) solution containing 10% galactose.</li>
	<li>Add, to a 250 mL flask, 0.04 g CSM-His, 0.335 g YNB + nitrogen, and water to 47.5 mL. Autoclave. Add 2.5 mL of 40% glucose to get 50 mL of CSM-His + YNB + 2% glucose.</li>
	<li>Prepare 1 L of 1M Tris pH 7.0 by mixing 121.14 g of Tris base with 800 mL of water. Add concentrated hydrochloric acid until the pH reaches 7.0. Add water to a final volume of 1 L and pass through a 0.2 &#181;m filter.</li>
	<li>Prepare 500 mL of 0.5 M ethylenediaminetetraacetic Acid (EDTA) pH 8.0 by mixing 73.06 g of EDTA with 400 mL water. Add sodium hydroxide pellets until the EDTA has dissolved and the pH reaches 8.0. Add water to a final volume of 500 mL and pass through a 0.2 &#181;m filter.</li>
	<li>Prepare 100 mL of 100 mM phenylmethanesulfonyl fluoride (PMSF) by mixing 1.74 g of PMSF with 200 proof ethanol. Store at -20 &#176;C.</li>
	<li>Prepare 225 mL of 2x bead wash buffer consisting of 50 mM Tris pH 7.0, 1.4 M NaCl, 20% glycerol, and 1 mM EDTA pH 8.0.</li>
	<li>Prepare 100 mL of the size exclusion chromatography buffer (S200 buffer) consisting of 20 mM 4-Morpholinoethanesulfonic acid hydrate (MES) pH 6.5, 100 mM NaCl, 2% glycerol, and 0.03% n-Dodecyl-beta-D-Maltopyranoside (DDM).</li>
	<li>Prepare 100 mL of membrane resuspension buffer consisting of 50 mM Tris pH 7.0, 500 mM NaCl, and 10% glycerol.</li>
	<li>Prepare 10 mL of a 3x SDS-PAGE loading dye by mixing 3 mL 20% sodium dodecyl sulfate (SDS), 3 mL glycerol, 2.4 mL 1M Tris pH 6.8, 0.03 g bromophenol blue, and 1.6 mL 2-mercaptoethanol.</li>
</ol>
2. Overexpression of Borate Transporter in S. cerevisiae
<ol>
	<li>Inoculate three transformed yeast colonies in 50 mL of CSM-His + YNB + 2% glucose media and shake overnight at 190 rpm at 30 &#176;C.</li>
	<li>The following day use a spectrophotometer to determine the optical density at a wavelength of 600 nm (OD600) and inoculate to an OD600 of 0.01 each of four 2 L flasks that each contain 500 mL of CSM-His + YNB + 2% glucose media.</li>
	<li>Shake at 190 rpm at 30 &#176;C for 30 h to allow the cells to consume all glucose and grow to a high density. 
	NOTE: A longer growth time results in larger cell yields, larger harvested membrane yields, and ultimately larger purified protein yields.</li>
	<li>Induce expression by adding 125 mL of 5x YP media supplemented with 10% galactose for a final induction concentration of 2% galactose. Shake at 190 rpm at 30 &#176;C for 16 h.</li>
	<li>Harvest cells by spinning at 4,000 x g for 15 min. Resuspend the cells in 100 mL cold water. 
	NOTE: At this point cells may be frozen at -80 &#176;C indefinitely, or the prep may continue into cell lysis and membrane harvesting. We typically harvest 40-45 g of cells.</li>
</ol>
3. Harvesting of Yeast Membranes
<ol>
	<li>Add to the cell resuspension 11.25 mL of 1M Tris pH 7.0, 0.45 mL of 0.5 M EDTA, and 2.25 mL of 100 mM PMSF, the latter two of which act as protease inhibitors. Add water to a final volume of 225 mL, which results in a cell resuspension buffer of 50 mM Tris pH 7.0, 1 mM EDTA, and 1 mM PMSF. 
	NOTE: If using frozen cells allow cells to thaw thoroughly, about 1 h at room temperature, because if they remain partially frozen, they will resist lysis by bead beating.</li>
	<li>Add the cell resuspension into a 450 mL metal canister for bead beating. Top off the remaining volume with cold 0.5 mm glass beads.</li>
	<li>Assemble a bead-beating chamber with the rotor and immerse in an ice bath. Perform six 1 min pulses, separated by 2 min rest periods to prevent overheating the lysate.</li>
	<li>Assemble a vacuum filtration apparatus by using a plastic disposable bottle top filter screwed into a glass bottle. Remove the filtration membrane, because the remaining plastic device can capture the beads while allowing lysate to pass. Separate the beads from the lysate by pouring the contents of the bead beating chamber onto the assembly while applying the vacuum.</li>
	<li>Wash the bead beating chamber with 225 mL of a 2x wash buffer that contains 50 mM Tris pH 7.0, 1 mM EDTA, 1 mM PMSF, 1.4 M NaCl, and 20% glycerol. Empty the contents of the chamber onto the beads to wash them. 
	NOTE: The wash gives a final volume of ~450 mL lysed cells and significantly increases harvested membrane yields. The final concentrations are 50 mM Tris pH 7.0, 1 mM EDTA, 1 mM PMSF, 10% glycerol, and 700 mM NaCl, and the primary purpose of elevating the salt concentration is to help dissociate peripheral membrane proteins.</li>
	<li>Spin down the cell lysate for 15 min at 15,000 x g. Pour the supernatant into polycarbonate bottles and spin for 1 h at 135,000 x g in an ultracentrifuge to collect membranes. 
	NOTE: If an ultracentrifuge is not available, membranes may be collected by spinning for 3 h at 53,000 x g, a force that is achievable in floor model centrifuges.</li>
	<li>Discard the supernatant and weigh the bottles with the membrane pellets. Resuspend the membrane pellets in approximately 35 mL membrane resuspension buffer containing 50 mM Tris pH 7.0, 500 mM NaCl, and 10% glycerol and add to a glass douncer. Weigh the empty centrifuge tubes to determine the mass of membranes harvested. 
	NOTE: In an efficient membrane harvest, the weight of membranes will be equal to approximately 20% the weight of cells used for that membrane harvest. This protocol gives typical cell yields of 40-45 g, and thus we likewise expect a yield of 8-9 g membranes.</li>
	<li>Dounce homogenize the membranes, and aliquot into 50 mL conical tubes that may now be stored indefinitely at -80 &#176;C. 
	NOTE: For this experiment, typically two aliquots are made, to allow two separate protein purifications to be performed from one original cell preparation.</li>
</ol>
4. Solubilization and Purification of Protein
<ol>
	<li>To a beaker add a stir bar and 150 mg of n-Dodecyl-&#946;-D-Maltopyranoside (DDM) per g membrane being used. Keep protein on the ice or at 4 &#176;C at all times. 
	NOTE: DDM is hygroscopic and should be stored in a glass jar with desiccant at -20 &#176;C when not in use. Also, it is typical to use between 4-5 g of membranes in a purification to obtain an appreciable amount of pure protein.</li>
	<li>Thaw membranes and bring to a final volume of 15 mL per g membrane in membrane resuspension buffer supplemented with 1 mM PMSF and 20 mM imidazole pH 8.0. Add membranes to the beaker with DDM and stir for 1 h at 4 &#176;C. 
	NOTE: The DDM concentration will be 1% w/v, but for the solubilization of membrane proteins it is more critical to be aware of the mass of detergent added per g membrane.</li>
	<li>Spin for 25 min at 135,000 x g at 4 &#176;C to pellet non-solubilized material. Filter the supernatant through a 5 &#181;m syringe filter.</li>
	<li>Load the sample by the peristaltic pump set to a flow rate of 1 mL/min onto a 1 mL immobilized nickel affinity column equilibrated in 20 mM Tris pH 7.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole pH 8.0, and 0.05% DDM. 
	NOTE: For lower expressing proteins, improved purity and yields were observed by using a 1 mL instead of 5 mL column, and nickel instead of cobalt ions for affinity chromatography.</li>
	<li>Wash the column with 10 column volumes of a wash buffer containing 20 mM Tris pH 7.0, 500 mM NaCl, 10% glycerol, 80 mM imidazole pH 8.0, and 0.05% DDM. 
	NOTE: The imidazole concentration that can be used during washes for a 10-His-tagged protein is higher than is commonly appreciated and results in improved purity.</li>
	<li>Elute the protein in the buffer containing 20 mM Tris pH 7.0, 200 mM NaCl, 10% glycerol, 300 mM imidazole pH 8.0, and 0.05% DDM. Collect the eluate in ten 1 mL fractions and run on a 4-20% Tris-glycine SDS-PAGE gel along with solubilized lysate and wash fractions.</li>
	<li>Pool peak fractions, usually about 5-6 mL, and concentrate to 500 &#181;L or less volume in a 50 kDa cutoff concentrator in a benchtop centrifuge refrigerated at 4 &#176;C. 
	NOTE: 500 &#181;L is a typical maximum volume injected into size exclusion chromatography (SEC) columns. At this point the protein may be stored indefinitely at -80 &#176;C or continue onto SEC.</li>
	<li>Filter the protein through a 0.2 &#181;m spin column filter and inject onto a size exclusion column (e.g., Superdex-200) equilibrated in S200 buffer: 20 mM Mes pH 6.5, 100 mM NaCl, 2% glycerol, and 0.03% DDM. 
	NOTE: For the subsequent glutaraldehyde cross-linking assay, the buffering agent must not contain a primary amine. SEC is an opportunity to exchange buffers if necessary, as done here when exchanging Tris for Mes.</li>
	<li>Run the peak fractions on a 4-20% Tris-glycine SDS-PAGE gel. Stain the gel, collect pure peak fractions, and concentrate in a 50 kDa-cutoff concentrator at 4 &#176;C.</li>
	<li>Determine the protein concentration by measuring the absorbance at a wavelength of 280 nm, and store indefinitely at -80 &#176;C.</li>
</ol>
5. Glutaraldehyde Cross-linking Assay
<ol>
	<li>Prepare a 10 &#181;L reaction by mixing 3 &#181;L of 0.5 mg/mL protein in S200 buffer, 5 &#181;L of S200 buffer, 1 &#181;L of either water or 20% sodium dodecyl sulfate (SDS), followed by 1 &#181;L of 1.5% glutaraldehyde. 
	NOTE: This will give a 1x reaction of 0.15 mg/mL protein and 0.15% glutaraldehyde. Samples containing a pre-treatment of SDS are important negative controls and&#160;should be mixed and incubated at room temperature for 5 min before adding glutaraldehyde.</li>
	<li>Incubate the reaction for 30 min at room temperature. Terminate the reaction by adding 5&#181;L of 3x SDS-PAGE gel loading dye, which contains an excess of Tris buffer that quenches the glutaraldehyde.</li>
	<li>Load all 15 &#181;L onto 4-20% Tris-glycine SDS-PAGE gel, which will load 1.5 &#181;g protein per lane. Run the gel at 200 V for 30 min. Stain and determine the extent of dimer cross-linking by evidence of a band that runs at twice the size of the denatured monomer. 
	NOTE: A glutaraldehyde titration and time course may be performed first to find the best conditions for a homomeric transporter.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Bor1+supports+an+elevator+transport+mechanism+for+SLC4+anion+exchangers.">Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (38), 10542-10546 (2016).</li><li>Coudray, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+SLC4+transporter+Bor1p+in+an+inward-facing+conformation.">Structure of the SLC4 transporter Bor1p in an inward-facing conformation.</a> Protein Science. 26 (1), 130-145 (2016).</li><li>Arakawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+anion+exchanger+domain+of+human+erythrocyte+band+3.">Crystal structure of the anion exchanger domain of human erythrocyte band 3.</a> Science. 350 (6261), 680-684 (2015).</li><li>Huynh, K. 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The authors have nothing to disclose.

Here we have shared detailed protocols that result in the purification to homogeneity of three distinct eukaryotic borate transporters. The protocols presented here are derived from other protocols for the expression of integral membrane proteins in S. cerevisiae1,14, and our optimizations result in both improved purity and improved yields. The parameters optimized here include cell culture growth volumes and times, bead-beating lysis procedures, buffer composition during cell lysis and protein purification, amount of detergent used per gram membrane, metal ion identify in affinity purification, volume of the affinity column, and the implementation of a cross-linking assay to evaluate homolog and detergent suitability by assessing the multimeric state of oligomeric transporters. The protocols are successful for multiple SLC4 homologs from the plant and fungal species. A limitation of all strategies for the purification of integral membrane proteins is that there exists no universal membrane protein purification method that is guaranteed to work. It is possible that our protocols are more likely to be successful for proteins closer in sequence identity and structure to SLC4 transporters, and thus members of the SLC4, SLC23, and SLC26 families could be promising targets15. Likewise, the more evolutionarily distant from the borate transporters a membrane transporter might be, the more likely the protocol will have to be different, such as by varying the expression system, detergent, or other key parameters4.
The protocol takes advantage of the most commonly used affinity tag in protein purification, the His-tag. Despite the presence of impurities in the initial eluted fractions, the combinations of nickel affinity, extensive washing, and subsequent SEC purification results in the highly purified protein. A 10-His-tag allows for the more stringent imidazole washes and thus can remove more background binding proteins than can be removed in washes permitted by 8-His- or 6-His-tags. Our selections for pure fractions err on the conservative side of the most highly pure gel fractions, which correspond to the peak SEC fractions. Final protein yields can be increased by pooling and concentrating more fractions, albeit with the trade-off of slightly less pure protein. The methods presented here enabled the purification of AtBor1 in quantities that led to the determination of its crystal structure7, with purification differences consisting of cutting off the His-tag and exchanging the transporter into a different detergent to improve crystal diffraction7.
Our protocol raises important considerations for the selection of homologs and the detergent used to solubilize and purify them. DDM is a common first choice for detergent because it is relatively mild, often successful at solubilizing, and has been used in structural and functional studies of a wide variety of membrane proteins. In determining whether a detergent is a poor selection for a membrane, a common method of evaluation is whether the protein gives a single monodisperse peak on an SEC chromatogram, rather than a large peak in the void volume or a range of polydisperse peaks, which indicate misfolded or unstable protein. A more subtle consideration is raised by our purification of ScBor1. It purifies in large quantities and looks favorable and monodisperse on an SEC chromatogram, which suggests it could be an attractive target for structural studies. However, our cross-linking assay reveals that it is a monomer when purified. While it is possible that ScBor1 could natively exist as a monomer&#160;in the cell, studies indicate that SLC4 transporters and their homologs are likely to be dimers7,8,9,10. Our development of the cross-linking assay occurred after crystallizing and solving the structure of AtBor1. However, had we been able to evaluate ScBor1 in comparison with AtBor1 with the cross-linking assay, valuable time and research efforts could have been redirected to the pursuit of AtBor1 instead of ScBor1, the latter of which ultimately was not successful in crystallization and diffraction experiments. This assay can thus help researchers distinguish between either homologs or detergents to use when pursuing structural studies in order to prioritize conditions in which the protein maintains its suspected native conformation. Additionally, the assay can be used to probe which amino acids are critical for multimerization, in order to find amino acid substitutions that destabilize the multimerization interface and lead to obligate monomers. Such an approach has been used to probe the functional significance of homomeric assembly in membrane transporters16,17,18.
A general advantage of our method is that yeast has been shown to enable the expression of many challenging membrane proteins of diverse function1. Additionally, its low cost and growth times promote its accessibility for many research efforts that may be less able to use expression strategies that require tissue culture or expensive growth media. The procedures presented here are relatively inexpensive and can be performed in one week which underscores the feasibility of the approach. Implementation of these protocols can help enable structural and functional studies of other challenging membrane proteins.

The difficulty in obtaining sufficient quantities of purified membrane protein remains a critical bottleneck in the pursuit of structural and functional studies of receptors, ion channels, and transporters. Many protocols exist for&#160;moderately high throughput pipelines to screen and find candidate membrane proteins that express well enough to enable subsequent in vitro studies1,2,3,4. Typically, proteins are tagged with an N- or C-terminal green fluorescent protein (GFP), and expression levels are monitored either by in-gel fluorescence or by fluorescence-detection size exclusion chromatography (FSEC)5. Such approaches enable the triaging of membrane protein candidates into high expressing proteins, moderate or low expressing proteins, or proteins that express poorly or not at all. This approach works well when the experimental design is to investigate large numbers of candidate genes with the intention of selecting whichever protein expresses the best. However, in some cases, an experimental approach is predicated on studying a particular membrane protein, which can be challenging when that protein&#39;s expression levels are in the moderate or low range. Additionally, sometimes in these cases, expression levels can be only minimally increased by altering the construct via truncations, thermostabilizing mutations, or codon optimization. It is, therefore, sometimes necessary to optimize membrane protein expression and purification protocols for membrane proteins that express only moderately well.
The SLC4 family of transporters includes the bicarbonate transporter Anion Exchanger 1 (also known as Band 3), the most abundant membrane protein in red blood cells6, and a key driver of cellular respiration. The SLC4 family is homologous with borate transporters, which are critical in plants and have been shown to exhibit a similar structure to Anion Exchanger 1 as well as to sodium-coupled SLC4 transporters7,8,9,10. Here we report optimized protein expression and purification protocols using S. cerevisiae to purify three different borate transporters found in yeast and plants. We highlight in detail key steps we took to optimize the yield and efficiency of the purifications to homogeneity. Additionally, we report a chemical cross-linking assay using glutaraldehyde to monitor the multimeric assembly of these transporters in the context of a purified protein-detergent-lipid complex. The cross-linking experiment can help evaluate homolog and detergent suitability by assessing the multimeric state of oligomeric transporters after purification.
This protocol assumes that the desired transporter has been cloned into a 2 &#181; derived plasmid under inducible control of the GAL1 promoter for expression in S. cerevisiae. For these procedures, DNA was used encoding full-length wild-type borate transporters Saccharomyces cerevisiae Bor1 (ScBor1)11, Arabidopsis thaliana Bor1 (AtBor1)12, and Oryza sativa Bor3 (OsBor3)13. The C-terminus in each construct is appended with a 10-His-tag and a thrombin cleavage site inserted between the transporter and the 10-His-tag to enable its removal if desired. The protocol will also assume that the plasmid has already been transformed into the DSY-5 expression strain of S. cerevisiae on complete supplemental selective media (CSM) lacking histidine.

<table border="0" cellpadding="0" cellspacing="0" style="width:823px;" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">2-mercaptoethanol</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:115px;">M3148</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Morpholinoethanesulfonic acid hydrate (MES)</td>
			<td style="width:199px;">Acros</td>
			<td>172591000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#196;kta Pure 25 L FPLC</td>
			<td>GE Healthcare</td>
			<td>29018224</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra 15 mL, 50 kDa MWCO concentrator</td>
			<td style="width:199px;">Millipore</td>
			<td>UFC905024</td>
			<td>for concentrating nickel column fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra 4 mL, 50 kDa MWCO concentrator</td>
			<td style="width:199px;">Millipore</td>
			<td>UFC805024</td>
			<td>for concentrating S200 fractions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Bacto Peptone</td>
			<td style="width:199px;">BD Diagnostics</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td style="width:199px;">BD Diagnostics</td>
			<td>288620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Glass Erlenmeyer flask, 2L</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>CLS44442L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Benchtop centrifuge</td>
			<td style="width:199px;">Sorvall</td>
			<td style="width:115px;">Legend RT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bottle top filter</td>
			<td style="width:199px;">Nalgene</td>
			<td>595-4520</td>
			<td>the membrane is removed and used to filter glass beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol blue</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>114391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Complete supplement mixture without histidine</td>
			<td>Sunrise Science</td>
			<td>1006-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Galactose</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>RDD016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof</td>
			<td style="width:199px;">Pharmco-Aaper</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel tank SDS-PAGE system</td>
			<td style="width:199px;">Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bead-beating cell disruptor</td>
			<td style="width:199px;">BioSpec</td>
			<td>1107900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass beads, 0.5mm</td>
			<td style="width:199px;">BioSpec</td>
			<td colspan="2">11079105&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Glutaraldehyde</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">16019</td>
			<td>sent as an 8% solution under nitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Glycerol</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">HiTrap IMAC FF column, 1 mL</td>
			<td style="width:199px;">GE Healthcare</td>
			<td style="width:115px;">17-0921-02</td>
			<td>charged with nickel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Hydrochloric acid</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imidazole</td>
			<td style="width:199px;">Acros</td>
			<td>12202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Instant Blue gel stain</td>
			<td style="width:199px;">Expedeon</td>
			<td style="width:115px;">ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">JA-10 rotor</td>
			<td style="width:199px;">Beckman</td>
			<td>369687</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">JA-14 rotor</td>
			<td style="width:199px;">Beckman</td>
			<td>339247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microcentrifuge tubes, 1.5mL</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td>3451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microcentrifuge, refrigerated</td>
			<td style="width:199px;">Fisher</td>
			<td>13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Protean Tris-glycine gels, 4-20%</td>
			<td style="width:199px;">Bio-Rad</td>
			<td>456-1096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minipuls 3 peristaltic pump</td>
			<td>Gilson</td>
			<td>F155005</td>
			<td>used for loading lysate onto affinity column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl-beta-D-Maltopyranoside (DDM)</td>
			<td style="width:199px;">Inalco</td>
			<td>1758-1350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nickel(II) Sulfate Hexahydrate</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>227676</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Orbital shaking incubator with temperature control</td>
			<td style="width:199px;">New Brunswick</td>
			<td style="width:115px;">C24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">p423 GAL1 plasmid with borate transporter insert</td>
			<td style="width:199px;"></td>
			<td style="width:115px;"></td>
			<td>available from authors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td style="width:199px;">Acros</td>
			<td>215740050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Polycarbonate ultracentrifuge tubes</td>
			<td style="width:199px;">Beckman</td>
			<td>355618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Polypropylene bottles, 250mL</td>
			<td style="width:199px;">Beckman</td>
			<td style="width:115px;">356011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Polypropylene bottles, 500mL</td>
			<td style="width:199px;">Beckman</td>
			<td>355607</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Precision Plus Protein Kaleidoscope Standards</td>
			<td style="width:199px;">Bio-Rad</td>
			<td>1610375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">S. cerevisiae expression strain DSY-5</td>
			<td style="width:199px;"></td>
			<td style="width:115px;"></td>
			<td>available from authors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium chloride</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium dodecyl sulfate</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:115px;">L3771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sodium hydroxide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:115px;">S8045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spin column with 0.2 &#181;m filter, 0.5mL</td>
			<td style="width:199px;">Millipore</td>
			<td>UFC30GV0S</td>
			<td>for filtering protein before injecting onto S200 column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sterile Falcon tubes, 15mL</td>
			<td style="width:199px;">Lab Depot</td>
			<td style="width:115px;">TLD431696</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sterile Falcon tubes, 50mL</td>
			<td style="width:199px;">Lab Depot</td>
			<td style="width:115px;">TLD431698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex 200 10/300 GL column</td>
			<td>GE Healthcare</td>
			<td>17-5175-01</td>
			<td>used for SEC purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filter, 5&#181;m</td>
			<td style="width:199px;">Pall</td>
			<td>4650</td>
			<td>for filtering lysate before loading affinity column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tris-base</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Type 70 Ti rotor</td>
			<td style="width:199px;">Beckman</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Ultracentrifuge</td>
			<td style="width:199px;">Beckman</td>
			<td style="width:115px;">L8-70M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YNB+Nitrogen without amino acids</td>
			<td>Sunrise Science</td>
			<td>1501-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression, solubilization, and purification of eukaryotic borate transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Typical gels for the eluted fractions from performing nickel affinity chromatography show all three proteins partially purified (Figure 1A). To the right of the ladder is the lysate, which in each case does not show a significant band corresponding to the borate transporter which is typical for proteins that do not overexpress well. The 80 mM wash lane shows minimal loss of 10-His-tagged protein despite the relatively high imidazole concentration. Bands corresponding to the borate transporters are readily apparent in eluted fractions, and fractions deemed to contain the bulk of the eluted protein are concentrated before injecting onto the S200 gel filtration column. At this stage, the proteins are insufficiently purified for many downstream applications, as indicated by extra bands in the concentrated sample before injecting onto the S200 column (Figure 1B). However, injecting the sample onto the size exclusion column reveals eluted fractions of high purity (Figure 1B-C). Chromatograms and their corresponding gels for each of the borate transporters are presented. Despite the moderate purity of the samples upon elution from affinity chromatography, the protein is highly pure upon eluting from the gel filtration column. Critically, the chromatograms reveal each protein to migrate primarily as a single monodisperse peak with little protein retained in the void volume. A stable and folded protein generally gives a monodisperse and symmetrical peak, while unstable, misfolded, or aggregated protein will generally give either multiple asymmetric peaks or a large peak in the void volume. It is typical to obtain a final yield of approximately 2 mg purified protein per L of yeast culture for ScBor1, and approximately 1 mg each of purified AtBor1 and OsBor3 per L culture. These numbers are the amount of protein purified from one aliquot of 4-5 g of membranes, which originates from one half of our original cell preparation.
The cross-linking experiment shows that purified homomeric transporters can have their assembly in solution readily assessed (Figure 2). The purpose of the samples containing a pre-treatment of SDS is to show that cross-linking is dependent on a folded state in solution and that cross-linking therefore does not occur when multimerization is disrupted by a harsh detergent. The results shown distinguish that one of the three borate transporters, ScBor1, is not dimerized when purified in DDM in these conditions, while the other two, AtBor1 and OsBor3, cross-link and show dimerization. It is possible to see that not all protein may cross-link. In this example, trace amounts of OsBor3 monomer are visible, while AtBor1 cross-links to completion. The extent of cross-linking will depend on the number of lysine residues in proximity to one another, and it is possible that other cross-linking reagents may efficiently cross-link different membrane proteins.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59166/59166fig1.jpg" /> 
Figure 1. Nickel affinity and size exclusion chromatography purification for three borate transporters. Each vertical panel (A), (B), and (C) contains data for ScBor1, AtBor1, and OsBor3. (A) Nickel affinity column. M is a ladder of molecular weight markers with weights in kDa specified to the left. L is the crude lysate, and W is the 80 mM imidazole wash. All other numbered lanes correspond to 1 mL fractions eluted with 300 mM imidazole. Bars above fractions indicate which were selected to be combined and concentrated for injections onto the column. (B) P indicates concentrated protein before injecting onto the S200 column. Eluted SEC fractions are to the right, with brackets used to match gel lanes with their chromatogram fractions in (C). The void volume is just after 8 mL. <a href="https://www.jove.com/files/ftp_upload/59166/59166fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59166/59166fig2.jpg" /> 
Figure 2. Cross-linking assay assesses purified transporters for multimeric assembly. A ladder with molecular weights is indicated on the left. Above all other lanes is shown which protein is present and whether the sample has had glutaraldehyde added or a pre-treatment of SDS before glutaraldehyde addition. Arrows indicate the positions of monomer and dimer for AtBor1 and OsBor3. ScBor1 is a smaller protein than AtBor1 and OsBor3 and runs as expected. <a href="https://www.jove.com/files/ftp_upload/59166/59166fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Expression, Solubilization, and Purification of Eukaryotic Borate Transporters at JoVE.com

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

Expression, solubilisation et Purification des transporteurs de Borate eucaryotes

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

expression-solubilization-purification-eukaryotic-borate

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JoVE Journal

Biochemistry

9.5K vues.  Davidson College. L’obtention de quantités suffisantes de protéines membranaires pour des applications en aval reste un défi technique important. Ce protocole permet la purification de plusieurs protéines de transport membranaire à l’homogénéité. Un avantage clé de cette technique est que Saccharomyces cerevisiae est un système d’expression eucaryotique rentable et rentable pour les protéines membranaires qui permet l’optimisation de nombreuses variables expérimentales clés.Commence...

Vidéo: Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

Thermostabilisation, Expression, purification et cristallisation du Serotonin Human Transporter Bound to<em&gt; S</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

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Biochimie

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

La purification d&#39;affinité en tandem de complexes protéiques à partir des cellules eucaryotes

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Expression, purification et activité antimicrobienne de S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

Expression et Purification des canaux ioniques de mammifères Bestrophin

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Expression et Purification de l’homme sensible lipides canal de Cation TRPC3 pour la détermination de structure par particule Cryo microscopie électronique

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Expression et purification de nuclease-Free Oxygen Sccatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

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.

Expression, Purification, Cristallisation et Essais enzymatiques des protéines contenant du domaine de l'hydrolase fumarylacetoacetate

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Caractérisation des transporteurs de membrane par l'expression hétérologue dans E. coli et la production de vésicules membranaires

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

Séparation mécanique et solubilisation protéique des sous-couches périphériques extérieures et intérieures des œufs de poule

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Mutagénèse par PCR, clonage, expression, protocoles de purification rapide des protéines et cristallisation du type sauvage et des formes mutantes de la tryptophane synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...