Name: purification of the cystic fibrosis transmembrane conductance regulator protein expressed in saccharomyces cerevisiae
Uploaded: 2014-05-10T15:00:00
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Description: Watch this Scientific Journal Video about Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae at JoVE.com

This work was funded by the US Cystic Fibrosis Foundation (CFF) through its CFTR 3D Structure Consortium. TR was funded by a UK CF Trust studentship, and NC by a UK BBSRC studentship. We acknowledge our colleagues in the CFF CFTR 3D structure consortium for their help and advice and for the design of the codon-optimized chicken CFTR sequence and purification tags.&#160;

1. Preparation of Buffers
<ol>
	<li>To make the 100x stock of protease inhibitor (PI) cocktail dissolve 96 mg AEBSF, 3.5 mg chymostatin, 10 mg E64, 16.5 mg leupeptin, 16.5 mg pepstatin, 348 mg PMSF, and 4 mg bestatin in 20 ml DMSO. Make 1 ml aliquots and store at -20 &#176;C. To make a 100x stock of benzamidine, dissolve 720 mg in 20 ml ultrapure water (ddH2O) and store in 1 ml aliquots at -20 &#176;C. This quantity is sufficient for one purification. In all buffers, PI and benzamidine stocks are used at a 1:100 dilution.</li>
	<li>Prepare &#8216;mPIB&#8217; (0.3 M Tris pH 8, 0.3 M sucrose, 2 mM DTT) and &#8216;CFTR&#8217; (50 mM Tris pH 8, 20% (v/v) glycerol, 1 mM DTT) buffers and chill to 4 &#176;C. Before use, add 1:100 of the protease inhibitor cocktail and 1:100 benzamidine according to the volume of mPIB used to resuspend the cell pellet (e.g., use 3.5 ml PI and 3.5 ml benzamidine in a total volume of 350 ml mPIB).</li>
	<li>Prepare solubilization buffers. Lyso-phosphatidyl glycerol-14 (LPG) solubilization buffer (50 mM Tris pH 8, 10% (v/v) glycerol, 50 mM NaCl, 1 mM DTT, protease inhibitors (PIs) and 4% (w/v) LPG) and dodecyl maltoside (DDM) solubilization buffer (50 mM Tris pH 8, 20% (v/v) glycerol, 1 M NaCl, 1 mM DTT, protease inhibitors, 4% (w/v) DDM). Buffer can be sonicated in a sonicator bath (35 W, 40 kHz) to assist with dispersal of the detergent, but avoid vortexing the mixture, as this creates bubbles. Chill to 4 &#176;C before use.</li>
	<li>CFTR purification buffer for the LPG purification is 50 mM Tris, 10% (v/v) glycerol, 50 mM NaCl, 1 mM DTT, 0.1% (w/v) LPG-14 and protease inhibitors. Prepare 350 ml of this buffer, and 150 ml of the same buffer plus 1 M imidazole. Adjust pH of both buffers to 8.</li>
	<li>The buffer for purification in DDM consists of 50 mM Tris pH 8, 20% (v/v) glycerol, 1 M NaCl, 1 mM DTT, 0.1% (w/v) DDM. Prepare 350 ml of this buffer, and 150 ml of the same buffer plus 1 M imidazole. Adjust pH of both buffers to 8.</li>
	<li>For gel permeation chromatography (GPC) buffer containing LPG, prepare 50 mM Tris pH 8, 10% (v/v) glycerol, 50 mM NaCl, 1 mM DTT, 0.05% (w/v) LPG-14. For GPC using DDM prepare a buffer of 50 mM Tris pH 8, 20% (v/v) glycerol, 1 M NaCl, 1 mM DTT, 0.1% (w/v) DDM. All buffers and ddH2O used on the GPC column should be filtered (0.2 &#956;m filter) and degassed before use.</li>
	<li>SDS-PAGE sample buffer (2x the working concentration): 50 mM Tris-HCl pH 7.6, 5% (v/v) glycerol, 5 mM EDTA, 0.02% (w/v) bromophenol blue. Make 700 &#956;l aliquots and store at -20 &#176;C. Before use, add 200 &#956;l of 20% (w/v) sodium dodecyl sulfate (SDS) and 100 &#956;l of fresh 0.5 M DTT. Incubate for at least 10 min with sample at room temperature before loading on gel. Do not heat; this will denature the GFP and may cause CFTR to aggregate.</li>
	<li>To make lipid stocks for reconstitution, dissolve a 4:1 (w/w) mixture of E. coli lipids and cholesterol in chloroform and methanol (2:1 v/v), and dry in a glass vial under N2 gas for 2 hr to form a lipid film. Add GPC buffer (with no NaCl) to a lipid concentration of 40 mg/ml and use repeated vortexing and sonication (35 W, 40 kHz) to clarify the solution.</li>
	<li>For the ATPase assay, prepare 100x stocks of ATPase inhibitors by dissolving SCH28080 to 1 mM in DMSO, NaSCN to 1 M in ddH2O and oligomycin to 2.5 mM in 100% (v/v) ethanol. Store in aliquots at -20 &#176;C. Make 100 ml of ATPase buffer with 50 mM Tris pH 7.4, 150 mM NH4Cl, 5 mM MgSO4 and 0.02% (w/v) NaN3. This can be stored at room temperature and used for several assays. Prepare a 5 mM ATP stock immediately prior to use and keep on ice. (N.B. Use Na2ATP to prevent excessive background signal from phosphate in the assay). Prepare the SDS stop solution (12% (w/v) SDS in ddH2O).</li>
	<li>For the Chifflet detection prepare buffer A (3% (w/v) ascorbate, 0.5% (w/v) ammonium molybdate, 0.5 M HCl) immediately before use and buffer B (2% (w/v) sodium citrate, 2% (w/v) sodium meta-arsenite, 2% (v/v) acetic acid).</li>
</ol>
2. Isolation of Yeast Microsomes
<ol>
	<li>S. cerevisiae expressing chicken CFTR are grown as described in O&#8217;Ryan et al. (2012)14. Store the material from a 20 L fermentation in two aliquots at -80 &#176;C for up to 6 months.</li>
	<li>Defrost one aliquot of cells rapidly and resuspend in 3 ml chilled mPIB per gram of cells.</li>
	<li>Disrupt cells in a bead mill using glass beads of 425-600 &#956;m diameter. Use five&#160;1 min periods of cell disruption separated by 1 min rest periods. (The rest periods are essential to ensure that the cells are not heated during disruption.)</li>
	<li>Monitor cell disruption by centrifugation of a 1 ml sample of the cell lysate from the bead mill. Centrifuge (12,000 x g, 4 &#176;C, 5 min) in a bench top centrifuge. Dilute the supernatant to 1:50 with mPIB in a cuvette and measure the A380. If A380 &#62; 0.1, or has stopped increasing despite several repeated bead-beating cycles, proceed to the following step. If not, repeat 2.3-2.4.</li>
	<li>Centrifuge the total cell lysate (12,000 x g, 4 &#176;C, 20 min). Retain the supernatant. Discard the pellet (containing unbroken cells and mitochondria), but if there is any doubt about the efficiency of cell breakage (see 2.4), then retain the pellet also.</li>
	<li>Centrifuge the supernatant from the previous step (200,000 x g, 4 &#176;C, 1.5 hr). Discard the supernatant and resuspend the pelleted microsomal membranes in CFTR buffer. If the microsomes are intended for purification using DDM, supplement the CFTR buffer with 1 M NaCl.</li>
	<li>Repeat the centrifugation of the resuspended membrane fraction (100,000 x g, 4 &#176;C, 1 hr) and discard the supernatant.</li>
	<li>Resuspend the pelleted microsomes in a minimum volume of CFTR buffer (final volume 5-15 ml, total microsomal protein 70-200 mg). A Bradford assay may be used to determine the total concentration of microsomal proteins25. In addition the fluorescence emission spectrum of the membranes should be measured (excitation = 485 nm, emission = 500-600 nm) and should have a distinct GFP fluorescence peak (maximum at 512 nm). CFTR can be specifically detected on an SDS-PAGE gel, scanned under GFP fluorescence conditions (Figure 1).</li>
	<li>Flash-freeze the resuspended microsomes by plunging into liquid nitrogen and store at -80 &#176;C, or continue to Step 3.</li>
</ol>
3. Solubilization of Microsomes
<ol>
	<li>If frozen, defrost microsomes immediately before use in a water bath set to 10 &#176;C.</li>
	<li>For the solubilization of membranes, dilute the microsomes with an equal volume of the relevant solubilization buffer (Step 1.3) to give a final detergent concentration of 2% (w/v) and a microsomal protein concentration 5 mg/ml. Incubate this mixture for 1 hr at 4 &#176;C with agitation (tube rotator). Retain 200 &#956;l for analysis.</li>
	<li>Centrifuge the mixture (100,000 x g, 4 &#176;C, 45 min). Remove the supernatant containing the solubilized membrane proteins, pass it through a 0.45 &#956;m syringe filter and store on ice. Measure the fluorescence of the supernatant (as in Step 2.8).</li>
	<li>Resuspend the insoluble fraction in 1% (w/v) SDS solution to a volume equal to the soluble fraction. Measure the fluorescence in this fraction and retain an aliquot of 50 &#956;l for SDS-PAGE analysis.</li>
</ol>
4. Nickel-affinity Purification of CFTR
<ol>
	<li>Link two 5 ml nickel sepharose columns in series. Wash with 2 column volumes (CV) 20% (v/v) ethanol, followed by 2 CV ddH2O, then wash the column with 2 CV of solubilization buffer (Step 1.4-1.5), containing 1 M imidazole. Repeat with 2 CV of solubilization buffer lacking imidazole.</li>
	<li>Add imidazole to a final concentration of 5 mM to the solubilized material (Step 2.8) and manually load the material onto the column or into a sample loop if using an automated liquid chromatography device.</li>
	<li>Load the solubilized material onto the column at a flow rate of 0.5 ml/min, and wash with 2 CV of imidazole-lacking buffer at the same flow rate to remove unbound material. Collect fractions in 50 ml Falcon tubes.</li>
	<li>For the first wash, use 3 CV of purification buffer with 40 mM imidazole at a flow rate of 1 ml/min. Collect 2 ml fractions.</li>
	<li>For the second wash, use 3 CV of purification buffer with 100 mM imidazole. Collect 2 ml fractions.</li>
	<li>Elute CFTR from the HisTrap column with 3 CV of purification buffer with 400 mM imidazole. Collect 2 ml fractions.</li>
	<li>Monitor fluorescence in eluted fractions (Step 2.8).</li>
	<li>Retain aliquots of peak fractions for SDS-PAGE analysis. Flash freeze remaining peak fraction samples and store at -80 &#176;C, or continue to the next purification step.</li>
</ol>
5. Gel Permeation Chromatography (GPC) Purification of CFTR
<ol>
	<li>Equilibrate the column (Superose 6 10/300 GL) with 1.2 CV ddH2O followed by 1.2 CV GPC buffer.</li>
	<li>During Step 5.1, concentrate the Ni-affinity purified fractions with the highest GFP fluorescence using a 100,000 MWCO centrifugal filter at 4 &#176;C. If purifying in DDM, avoid concentrating the sample above a protein concentration of 0.3 mg/ml protein as this will cause significant sample loss. Remove the retentate from the concentrator and centrifuge at 100,000 x g for 30 min at 4 &#176;C to pellet large particles.</li>
	<li>Inject this sample onto the column and elute with an isocratic gradient of 1.2 CV GPC buffer. Collect 0.5 ml fractions.</li>
	<li>Measure GFP fluorescence as in section 2.8 to identify those fractions containing CFTR. Retain a small volume (e.g. 50 &#956;l) of each for analysis by SDS-PAGE.</li>
	<li>Freeze fractions in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
6. Reconstitution of CFTR
<ol>
	<li>Add lipids (Step 1.8) to the purified CFTR at lipid-to-protein ratio 100:1 (w/w) and incubate at 4 &#176;C for 1 hr. Similarly set up a lipid-only control, substituting the purified protein with the same volume of GPC buffer.</li>
	<li>Remove detergent from the protein/lipid mixture using hydrophobic adsorbent beads. Wash adsorbent beads in 5 CV ddH2O, 5 CV 70% (v/v) ethanol, 5 CV ddH2O, and 5 CV GPC buffer lacking the detergent. Add 200 mg of washed adsorbent beads per ml of purified protein and incubate at 4 &#176;C overnight with gentle agitation.</li>
	<li>Collect the reconstitution sample from the adsorbent beads into a fresh tube using a thin-ended pipette tip.</li>
</ol>
7. Measurement of ATPase Activity
<ol>
	<li>Determine the rate of CFTR-specific ATPase activity using a modified Chifflet assay26,27 in a 96-well plate format. With sodium phosphate stock solution (0.65 mM) prepare 0-20 nmol phosphate in a final volume of 50 &#181;l as standards. Use a 1:1 mixture of CFTR buffer and ATPase buffer to dilute the phosphate stock.</li>
	<li>Incubate both reconstituted CFTR and blank liposomes with 1:100 (v/v) ATPase inhibitors (Step 1.9) on ice for 10 min. Use at least 5 &#956;g of reconstituted CFTR.</li>
	<li>Add ATP (Step 1.9) to a final concentration of 2 mM and incubate at 25 &#176;C for 1 hr. Stop the reaction by adding 40 &#956;l of 10% (w/v) SDS (Step 1.9) to each well (including the standards).</li>
	<li>Add 100 &#956;l of buffer A (Step 1.10) and incubate for 10 min. Add 100 &#956;l buffer B (1.10) to each well and measure the absorbance at a wavelength of 800 nm in a 96-well plate-compatible UV/Vis spectrophotometer.</li>
	<li>Convert absorbance at 800 nm into an amount of liberated phosphate using the phosphate standards. Calculate the rate of ATP hydrolysis after subtracting background signal (liposome-only wells).</li>
	<li>For non-reconstituted CFTR follow the same protocol using CFTR buffer for the background readings.</li>
</ol>

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The authors have no competing financial interests nor other conflicting interests with respect to this work.

We have previously described a method for the overexpression of murine CFTR14. Since the publication of that protocol, we have expressed and purified several different orthologs of CFTR using the same system. All orthologs tested so far purified well in the LPG detergent, whilst the DDM purification showed more variation across different orthologs (data not shown). This flexibility illustrates the strength of the yeast approach: it is possible to screen many constructs with relative rapidity in order to select one for a particular purpose.
Washing the yeast microsomes with buffer containing 1 M NaCl prior to solubilization with DDM results in a cleaner microsome preparation and reduces contaminants at later stages. This step is unnecessary in the LPG protocol as the final CFTR sample is &#62;90% pure without the microsome wash. Furthermore, purification in DDM requires several alterations to the buffers for solubilization and purification, namely the addition of extra glycerol and salt. Together, these additions considerably increased the binding of the DDM-solubilized protein to the column.
The DDM purification methodology has scope for improvement, in particular the removal of a 40 kDa major contaminant that, judged by mass spectrometry, is due to the yeast ribosomal subunit L3, which appears to have an inherent affinity for the nickel resin. There is no obvious polyHis sequence in the L3 protein, but examination of its 3D structure when bound to the ribosome (PDB = 1FFK) shows that the folded L3 subunit has a potential polyHis cluster. That this band is less problematic in LPG-purified material may be due to the harsher LPG detergent.
Though the purification in DDM appears to be poorer than that in LPG, milder detergents such as DDM may be more compatible with functional and structural analyses and have already been used in several X-ray crystallographic studies of membrane proteins15-21. Furthermore, our results indicated that the use of LPG leads to loss of ATPase function in CFTR relative to purification in DDM. Hence we would recommend the LPG-based purification protocol for the generation of CFTR where the purity is crucial, for example in applications such as the characterization of post-translational modifications, or in the generation of antibodies, the LPG-based protocol would be chosen. On the other hand in applications where the activity and fully native state of the protein is essential, we would propose the DDM-based protocol as a better option.
To conclude, this protocol describes a reproducible method for the isolation of CFTR in the zwitterionic detergent LPG-14 or the non-ionic detergent DDM. As such it indicates a greater range of purification conditions for CFTR than have previously been reported10-13. In addition milligram quantities of purified CFTR can be obtained using these procedures when combined with a high volume yeast growth system such as a 20 L fermenter and a high capacity cell harvesting system such as a 6 L low speed centrifuge rotor. The CFTR obtained has a cleavable GFP tag which allows easy monitoring of the protein in various biochemical and biophysical assays.
The reagent described in this manuscript (chicken CFTR&#8211;containing plasmid or frozen yeast cells) can be obtained through the Cystic Fibrosis Foundation (USA).

Cystic fibrosis (CF) is the most common genetic disorder in Europe and North America with an incidence of about 1 in 2,500 live births. CF occurs when mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause loss of its function at the plasma membrane of epithelial cells1. The most serious consequence of this defect is irreversible lung damage, which shortens the life expectancy of sufferers to &#60;40 years of age2,3.
CFTR is an ATP-binding cassette (ABC) transporter that has evolved to become an ion channel1,4. Despite its quite altered function in the plasma membrane of cells, it still retains significant sequence homology with other ABC transporters. Intriguingly, the specialized parts of CFTR (i.e. its regulatory region and its N- and C-termini) share no significant sequence similarity with other metazoan ABC transporters, hence there are no clues as to the origins of these sequences in CFTR.&#160; On the basis of its primary structure, CFTR is classified as a C-family member of the ABC transporter family, but there is no strong evidence for a residual functional linkage to this sub-family. There have been some reports of glutathione transport activity for CFTR5-7, which would be consistent with the roles of other C-family members8,9, although other reports suggest that reduced glutathione may inhibit the CFTR ATPase activity, rather than showing the substrate-induced stimulation that characterize the ABC transporters10. Measurement of ion conductance is sufficiently sensitive to allow the channel activity of single CFTR molecules to be studied1 and CFTR channel properties have been monitored as a function of time, temperature, ATP concentration, membrane potential, and phosphorylation state, as well as in the presence of a host of small molecule inhibitors, potentiators, and modifiers. These studies have also added significantly to our knowledge of how ABC transporters function. Nevertheless, expression of CFTR in significant amounts and its subsequent purification has proven to be particularly challenging and success has been limited to a few laboratories10-13.
The need to develop more effective drugs is pressing, yet this process has been hindered by the lack of purified CFTR for screening small molecules. Solving the CFTR expression and purification problem would enable high-throughput drug screening aimed at correcting the primary defect in CF and would also open up a route for high-resolution structural studies to inform rational drug design. Moreover, even relatively basic biochemical characteristics of the protein, such as its functional oligomeric state, interacting proteins and ATPase activity remain poorly characterized. We have previously reported a protocol for the large-scale expression of GFP- and His-tagged murine CFTR in S. cerevisiae14 and now further describe protocols for the purification of CFTR. We have used these methods to purify five orthologues of CFTR, and present data for the purification of chicken CFTR as an example. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification. Dodecyl-&#946;-D-maltoside (DDM) is a nonionic detergent that has been extensively used for both structural and functional studies of membrane proteins15-21. The ionic detergent 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14) is highly efficient in the solubilization of CFTR and has previously been used in the purification of functional membrane proteins10,22,23, including purification of CFTR from S. cerevisiae24.

<table border="0" cellpadding="0" cellspacing="0" style="width:492px;" width="491">
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		<col />
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2&#956;M syringe filter&#160;</td>
			<td style="width:115px;">Sartorius</td>
			<td style="width:161px;">FC121</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 kDa MWCO centrifugal concentrator (PES membrane)</td>
			<td style="width:115px;">Vivaspin</td>
			<td style="width:161px;">VS0641</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2ml microfuge tubes</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:161px;">72.695</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40Ti rotor</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;"><a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901">337901</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50ml Sterile Falcon Tubes</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:161px;">62.547.254</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Adenosine triphosphate disodium salt (Na2ATP)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A26209&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid chromatography system</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">28-4062-64</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aminoethylbenzenesulfonyl fluoride (AEBSF)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A8456</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bead-beating cell disrupter</td>
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			<td style="width:161px;">1107900</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge&#160;</td>
			<td style="width:115px;">HERMLE</td>
			<td style="width:161px;">Z300</td>
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			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge&#160;</td>
			<td style="width:115px;">Eppendorf</td>
			<td style="width:161px;">5417R</td>
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			<td height="21" style="height:21px;width:216px;">Benchtop microfuge&#160;</td>
			<td style="width:115px;">Fisher</td>
			<td style="width:161px;">13-100-511</td>
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			<td height="21" style="height:21px;width:216px;">Benzamidine hydrochloride</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">434760</td>
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			<td height="42" style="height:42px;width:216px;">Hydrophobic Beads SM-2 Adsorbent</td>
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			<td style="width:161px;">152-3920</td>
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			<td height="21" style="height:21px;width:216px;">Bromophenol blue</td>
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			<td style="width:161px;">114391</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge tubes</td>
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			<td style="width:161px;">357000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel imaging system</td>
			<td style="width:115px;">BioRad</td>
			<td style="width:161px;">170-808</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">C8667</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chymostatin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">C7268</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">D8418</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol (DTT)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">43815</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E.coli total lipid extract</td>
			<td style="width:115px;">Avanti lipids</td>
			<td style="width:161px;">100500</td>
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			<td height="42" style="height:42px;width:216px;">Epoxysuccinyl-leucylamido-butane (E-64)</td>
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			<td style="width:161px;">E3132</td>
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			<td height="21" style="height:21px;width:216px;">Glass beads, acid washed</td>
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			<td style="width:161px;">G8772</td>
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			<td height="21" style="height:21px;width:216px;">Glycerol</td>
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			<td style="width:161px;">65017</td>
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			<td height="21" style="height:21px;width:216px;">HisTrap HP columns (5 ml)</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-5247-05</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapid Coomassie Stain</td>
			<td style="width:115px;">Novexin</td>
			<td style="width:161px;">ISB1L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge JA-17 rotor</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">369691</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leupeptin</td>
			<td style="width:115px;">Merck</td>
			<td style="width:161px;">108975</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lyso-phosphatidyl glycerol-14 (LPG)</td>
			<td style="width:115px;">Avanti lipids</td>
			<td style="width:161px;">858120</td>
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			<td height="25" style="height:25px;width:216px;">MgSO4</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">M7506</td>
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			<td height="21" style="height:21px;width:216px;">Gel tank SDS-PAGE system</td>
			<td style="width:115px;">BioRad</td>
			<td style="width:161px;">165-8006</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">n-dodecyl-&#946;-D-maltopyranoside (DDM)</td>
			<td style="width:115px;">Affymetrix</td>
			<td style="width:161px;">D310S</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S6191</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaN3</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S2002</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">A9434</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligomycin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">75351</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultracentrifuge</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">392050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prestained protein standards</td>
			<td style="width:115px;">Fermentas</td>
			<td style="width:161px;">SM1811</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Desalting columns (Sephadex G-25)</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-0851-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pepstatin A</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">P4265</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenylmethanesulfonylfluoride (PMSF)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">P7626</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sch28080</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">S4443</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">L37771</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiocyanate (NaSCN)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:161px;">251410&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel filtration 10/300 GL column</td>
			<td style="width:115px;">GE Healthcare</td>
			<td style="width:161px;">17-5172-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-base</td>
			<td style="width:115px;">Formedium</td>
			<td style="width:161px;">TRIS01</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultracentrifuge tubes</td>
			<td style="width:115px;">Beckman Coulter</td>
			<td style="width:161px;">355618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex mixer</td>
			<td style="width:115px;">Star Labs</td>
			<td style="width:161px;">N2400-0001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic water bath</td>
			<td style="width:115px;">Ultrawave</td>
			<td>F0002202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multimode plate reader</td>
			<td style="width:115px;">BioTek</td>
			<td style="width:161px;">BTH1MF</td>
		</tr>
	</tbody>
</table>

purification of the cystic fibrosis transmembrane conductance regulator protein expressed in saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

The protocol described above is an efficient means to isolate CFTR-enriched microsomes, with almost complete recovery of CFTR during the cell breakage and preparation of the crude microsomes (Figure 1). Other cell breakage methods may also be employed effectively. We have utilized a French pressure cell, and other high-pressure/cavitation devices (also in combination with impacting against a ruby target) with equal efficiency. For convenience and low initial cost of the equipment, we find the bead-beating method the best.
Using LPG to solubilize and purify CFTR yielded 80 &#956;g protein/L culture at &#62;90% purity (Figure 2). The high yield was due to efficient solubilization of CFTR by LPG (compare Figure 2b, lanes 2 and 4). In addition, efficient and tight binding to the column resulted in minimal loss of CFTR in the unbound fraction and the absence of CFTR in the wash fractions (Figure 2, lanes 3, 5, and 6). The eluted protein had a purity of &#62;90%, estimated by Coomassie-stained SDS-PAGE gels and using densitometry of the CFTR and contaminant bands. Gel permeation chromatography (GPC) separated LPG-purified CFTR from low-molecular weight contaminants (Figure 4, lower panel).
The protocol for CFTR purification using DDM gives purity of about 60% and yield of roughly 50 &#956;g/L (Figure 3). Electron microscopy (EM) of negatively stained fractions from the GPC eluting at about 10 ml (Figure 4) showed that DDM-purified CFTR contains aggregates of 20-30 nm diameter as well as smaller particles of 10 nm diameter (data not shown). It is possible that the small aggregates can reversibly associate and dissociate as ultrafiltration with a 1 MDa cut-off filter failed to remove the EM-detectable aggregates. LPG-purified material did not adsorb to a glow-discharged grid, hence was studied by cryo-EM of unstained fractions. This showed a very homogeneous particle population of a relatively small size (6-8 nm diameter, data not shown).
Finally, the ATPase activity of the purified proteins was measured (Figure 5). As a member of the ABC protein family, CFTR has two nucleotide-binding domains (NBDs) capable of binding and/or hydrolyzing ATP. The data indicate that the purified protein was not able to hydrolyze ATP in the LPG-solubilized state and showed weak ATPase activity in the presence of DDM (Figure 5, unfilled bars). After the addition of lipids, and detergent removal, ATPase activity was 4-fold higher for samples that had been purified in DDM (13 nmol ATP/min/mg protein). The addition of lipids and removal of LPG similarly restored activity to CFTR that had been isolated using LPG, but with a final lower rate (1.5 nmol ATP/min/mg protein) than the DDM-purified and reconstituted material.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig1highres.jpg" src="/files/ftp_upload/51447/51447fig1.jpg" /> 
Figure 1.&#160;Monitoring levels of chicken CFTR in cell lysate (CL), supernatants (S) and pellets (P) during various centrifugation steps used for microsome isolation and washing. SDS-PAGE gels were visualized using the in-gel fluorescence of the GFP tag. The supernatant after cell breakage and centrifugation at 14,000 x g contains virtually all the CFTR (including degradation products).&#160; Ultracentrifugation at 200,000 x g sediments all the full-length CFTR leaving some fragments in the supernatant. Ultracentrifugation at 100,000 x g of salt-washed microsomes pellets nearly all the CFTR with the removal of some further fragments.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig2highres.jpg" src="/files/ftp_upload/51447/51447fig2.jpg" /> 
Figure 2.&#160;Purification of chicken CFTR in LPG by immobilized metal ion affinity chromatography.&#160; Fractions were analyzed by SDS-PAGE followed by Coomassie staining (upper panel) and fluorescence detection of the GFP tag (lower panel). Tracks: (1) Microsomes. (2) LPG-solubilized microsomes. (3) Unbound material. (4) Insoluble material. (5) &#38; (6) 40 and 100 mM imidazole washes. (7) Material eluted with 400 mM imidazole.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig3highres.jpg" src="/files/ftp_upload/51447/51447fig3.jpg" /> 
Figure 3.&#160;Purification of chicken CFTR in DDM by immobilized metal ion affinity chromatography. Fractions were analyzed by SDS-PAGE followed by Coomassie staining. The left hand panel shows fractions prior to elution. Several consecutive elution fractions are shown in the right hand panel with CFTR indicated by the arrow. Later fractions are enriched in a 40 kDa contaminant, which has been identified by mass spectrometry as ribosomal protein L3.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig4highres.jpg" src="/files/ftp_upload/51447/51447fig4.jpg" /> 
Figure 4.&#160;Purification of chicken CFTR by gel permeation chromatography. CFTR purified by Ni-affinity chromatography was concentrated and applied to a GPC column. The elution profile for CFTR (upper panel) purified in buffer containing LPG-14 (solid line) or DDM (dashed line) are overlaid. SDS-PAGE (lower panel) revealed that CFTR eluted between 8 and 11 ml.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51447/51447fig5highres.jpg" src="/files/ftp_upload/51447/51447fig5.jpg" /> 
Figure 5.&#160;ATPase activity of purified chicken CFTR fractions. Protein purified in DDM or LPG was assayed using a modified Chifflet assay26 in the presence of a cocktail of ATPase inhibitors to eliminate any background ATPase activity from F-, P- and V-type ATPases (unfilled bars). The rate of ATP hydrolysis was also measured after detergent removal and lipid addition (filled bars). The plot shows the mean and standard deviation (n=3). Differences between mean values for ATPase activity in presence and absence of lipid, and difference between activity in DDM and LPG are significant to p&#60;0.05.

Watch this Scientific Journal Video about Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae at JoVE.com

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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Biology

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...