Name: expression and purification of the cystic fibrosis transmembrane conductance regulator protein in saccharomyces cerevisiae
Uploaded: 2012-03-10T14:00:00
Duration: 14 min 56 s
Description: Watch this Scientific Journal Video about Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae at JoVE.com

We thank the Cystic Fibrosis Foundation (CFF) for funding this work through its CFTR 3D Structure Consortium (grant number FORD08XX0). We acknowledge the huge contribution of all our colleagues within the Consortium to this work, in particular in the design of the CFTR genes. We also acknowledge the invaluable contributions of Drs. James Birtley (NCSR Demokritos, Greece), Mark Young (University of Cardiff, UK) and David Drew (Imperial College, London, UK) in the early stages of the work. We especially thank Dr. Ina Urbatsch (Texas Tech. University, Lubbock) for critical reading of the manuscript.

1. Preparation of Media and Buffers
<ol>
	<li>YNB: For one litre, suspend 6.9 g yeast nitrogen base without amino acids and 0.77 g complete supplement mixture without uracil in 1 l water. Autoclave. Store at 4 &deg;C.</li>
	<li>YNBA: For 400 ml, suspend 2.76 g yeast nitrogen base without amino acids, 0.38 g complete supplement mixture without uracil and 8g bacteriological agar in 350 ml water. Autoclave. Mix 8 g glucose with 50 ml water and heat gently until dissolved. Sterilise through a 0.2 &mu;m filter and add to the YNBA whilst the agar is molten. Store at room temperature.</li>
	<li>20% glucose medium: For 500 ml, mix 100 g glucose with 500 ml YNB and heat gently until dissolved. Pass through a 0.2 &mu;M filter into a sterile Duran bottle. Store at room temperature.</li>
	<li>20% galactose medium: For 2 litres, mix 400 g galactose with 2l YNB and heat gently until dissolved. Pass through a 0.2 &mu;m filter into a sterile Duran bottle. Store at room temperature.</li>
	<li>Protease inhibitor stocks: CFTR is highly susceptible to proteolysis4. The authors found the following inhibitors to be effective in limiting proteolysis, though readers may wish to tailor this list to meet their own requirements. Store in 100 &mu;l aliquots at -20 &deg;C to reduce freeze-thaw problems. All inhibitors should be diluted from the stock solutions to the working concentration as below:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Inhibitor</td>
			<td>Stock Concentration </td>
			<td>Stock Preparation</td>
			<td>Working Concentration</td>
		</tr>
		<tr>
			<td>AEBSF</td>
			<td>200 mM</td>
			<td>Dissolve 48mg in 1ml distilled water</td>
			<td>0.2 mM</td>
		</tr>
		<tr>
			<td>Benzamidine</td>
			<td>300 mM</td>
			<td>Dissolve 36mg in 1ml distilled water</td>
			<td>0.3 mM</td>
		</tr>
		<tr>
			<td>Chymostatin</td>
			<td>4 mM</td>
			<td>Dissolve 2.5mg in 1ml dry DMSO</td>
			<td>4 &mu;M</td>
		</tr>
		<tr>
			<td>E-64</td>
			<td>7 mM</td>
			<td>Dissolve 2.5mg in 1ml distilled water</td>
			<td>7 &mu;M</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>20 mM</td>
			<td>Dissolve 10mg in 1ml distilled water</td>
			<td>20 &mu;M</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>15 mM</td>
			<td>Dissolve 10mg in 1ml dry DMSO</td>
			<td>15 &mu;M</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>1 M</td>
			<td>Dissolve 174mg in 1ml dry DMSO</td>
			<td>1 mM</td>
		</tr>
	</tbody>
</table>
<ol start="6">
	<li>DTT (1 M): Dissolve 154 mg dithiothreitol in 1ml distilled water. Store at -20 &deg;C. Use at 1:1000 dilution in buffers indicated.</li>
	<li>EDTA (0.5 M): Mix 29.22 g ethylenediaminetetraacetic acid with 100 ml water. Add 10 N NaOH dropwise until all of the EDTA has dissolved and the pH reaches 8. Make up to 200 ml with water and pass through a 0.2 &mu;m filter into a sterile Duran bottle. Store at room temperature.</li>
	<li>CRB (300 mM Tris-HCl, pH 7.4, 0.56 M sorbitol, 1 mM EDTA): Dissolve 18.17 g Tris-base in 350 ml water and adjust pH to 7.4 by addition of HCl. Add 51.35 g sorbitol and make volume up to 500 ml with water. Autoclave. Add 1 ml EDTA stock solution and store at 4 &deg;C.</li>
	<li>CFTR buffer (50 mM Tris, pH 8.0, 10% v/v glycerol): Dissolve 6.06 g Tris-base in 500 ml water. Adjust pH to 8 with HCl, add 100 ml glycerol and make up to 1 L with water. Autoclave and store at 4 &deg;C.</li>
	<li>2x load dye: 50 mM Tris-HCl (pH 7.6), 5% glycerol, 5 mM EDTA (pH 8.0), 0.02% bromophenol blue, 4% SDS, 0.05 M DTT. Make 10 ml, aliquot and store at -20 &deg;C.</li>
</ol>
2. Screening Transformants
This protocol assumes that the CFTR-GFP-8His fusion gene has been inserted into a yeast plasmid downstream to a GAL1 galactose promoter (Fig.1) and that the plasmid has been transformed into FGY217 cells, a Pep4 deletion mutant of S.cerevisiae10. Cells can be grown on YNBA plates and stored for several weeks at 4 &deg;C. For longer term storage, glycerol stocks should be made and stored at -80 &deg;C. Methods for cloning and transformation are described in detail by Drew et al. (2008)10.
<ol>
	<li>Pick 5-10 well-separated colonies from a transformation plate. Transfer each colony to a separate sterile 50 ml Falcon tube containing 9 ml YNB and 1ml of 20% glucose medium. For this step, it is important to have a final concentration of 2% glucose (w/v) in the culture to maintain cell growth. Grow overnight for 16 hours at 250 rpm, 30 &deg;C in an orbital shaking incubator.</li>
	<li>Make glycerol stocks for each of the screened colonies. Aseptically add 0.8 ml of the overnight cultures to 0.2 ml sterile glycerol in labeled screw-top vials, vortex briefly and store at -80 &deg;C.</li>
	<li>Dilute the remaining overnight cultures to a final volume of 50 ml in YNB, including 250 &mu;l of 20% glucose medium. For this step, it is important to dilute the glucose concentration to approximately 0.1% (w/w) in the culture because high glucose can repress the GAL1 promoter10. Grow cultures in labeled 250 ml Erlenmeyer baffled flasks to an OD600 of 0.7-0.8 at 250 rpm, 30 &deg;C in an orbital shaking incubator.</li>
	<li>Induce the cultures by addition of 5 ml 20% galactose medium to each flask and grow on for 16 hours.</li>
	<li>Confirm the expression of CFTR using fluorescence microscopy. Take 100 &mu;l of culture and add 100 &mu;l glycerol to limit cell mobility in solution. Analyse cells on a Delta Vision RT restoration microscope (or similar), using a blue laser under a FITC filter (excitation wavelength of 490 nm and emission wavelength of 528-538 nm). Positive expression of the CFTR-GFP fusion protein should be visible as a ring of fluorescence at the plasma membrane of the yeast cells. Untransformed yeast cells may be used as a control.</li>
	<li>Transfer the cultures into 50ml Falcon tubes. Harvest the cells by centrifugation at 3500 x g, 4 &deg;C for 10 minutes in a bench top centrifuge. Whilst the centrifuge is running, prepare 2 ml microfuge tubes with screw tops containing approximately 500 &mu;l acid-washed glass beads and place on ice. Discard the supernatants and resuspend each pellet in 500-800 &mu;l ice-cold CRB with protease inhibitors. Transfer the suspensions to the microfuge tubes containing the beads and keep on ice.</li>
	<li>Lyse the cells by vigorously shaking/vortexing each microfuge tube for 10 periods of 30 seconds, resting on ice in between periods. A beadbeater can be employed as an alternative, e.g. a BioSpec mini beadbeater operated for 3 min.</li>
	<li>Place the tubes into a benchtop microfuge and centrifuge at 3,500 x g, 4 &deg;C for 5 minutes. Transfer the supernatants containing the crude membrane population to clean microfuge tubes and place on ice. Add 500 &mu;l fresh ice-cold CRB with protease inhibitors to each tube and repeat the process to accumulate the membranes.</li>
	<li>Collect the crude membranes by spinning at maximum speed, 4 &deg;C in a benchtop microfuge for 2 hours. Discard the supernatant and resuspend each pellet in 50 &mu;l ice cold CFTR buffer.</li>
	<li>In clean microfuge tubes, mix 15 &mu;l of each suspension with 15 &mu;l 2x load dye by pipetting up and down. Incubate at room temperature for a 10 minutes. Do not boil the samples, as this will cause CFTR and other membrane proteins to form SDS-insoluble aggregates and also denature the GFP tag.</li>
	<li>Load the samples along with PageRuler Plus prestained protein standards (Fermentas) onto a 4-20% Tris/glycine gradient gel (NuSep) and run at 150 V for 40 minutes or until the dye-front is at the bottom of the gel.</li>
	<li>Identify the highest expressing cells by in-gel fluorescence. Place into a fluorescence imaging system such as a Typhoon scanner. Scan the gel using the blue laser at an excitation wavelength of 488 nm and an emission wavelength of 526 nm. The CFTR-GFP fusion should be visible at approximately 220 kDa. There will also be a weak fluorescent band visible at about 70 kDa which is probably an intrinsic yeast FAD-containing membrane protein (such as succinate dehydrogenase subunit A) 11,12.</li>
	<li>Stain the gel with Coomassie, destain and scan the gel for comparison to the fluorescence scan using a convenient image viewing software package. The Coomassie-stained gel allows a relative assessment of CFTR-GFP expression levels in different clonal lines after normalization for the amount of total protein loaded onto each track of the gel.</li>
	<li>Streak out the highest expressing cell line from its glycerol stock (2.2) onto a fresh YNBA plate and incubate at 30 &deg;C for 2-3 days. This plate may then be stored for up to 2 weeks at 4 &deg;C.</li>
</ol>
3. Large-scale Fermenter Culture
<ol>
	<li>Prepare pre-cultures for the fermenter. Scrape a 1cm2 area of cells from the YNBA plate (2.13) using a sterile loop and add to 45 ml YNB and 5 ml 20% glucose medium, such that the OD600 is approximately 0.1. Grow in 250 ml Erlenmeyer baffled flasks at 250 rpm, 30 &deg;C in an orbital shaking incubator until the OD600 reaches 1.</li>
	<li>Split the culture between two 2 l Erlenmeyer baffled flasks each containing 450 ml YNB and 25 ml 20% glucose medium. Grow these on at 250 rpm, 30 &deg;C in an orbital shaking incubator until the OD600 reaches 1.2.</li>
	<li>Whilst these pre-cultures are growing, set up the fermenter. Make 11.2l of YNB as described in 1.1, but dissolve an additional 8.28 g YNB and 0.95 g drop out supplement to compensate for the addition of glycerol at induction. Aseptically add the 11.2 l YNB and 75 ml 20% glucose medium to a sterile 20l fermenter vessel and adjust the running temperature to 30 &deg;C.</li>
	<li>Aseptically add the precultures to the fermenter and set the stirring speed to approximately 800 rpm and maintain the temperature at 30 &deg;C. Compressed air should flow at approximately 15 dm3min-1.Once the fermenter culture reaches an OD600 of 1.2, induce by aseptically adding 1.5 l YNB 20% galactose solution and 1.2 l glycerol. Reduce the temperature to 25 &deg;C and grow the culture for 16 hours.</li>
	<li>Transfer the fermenter contents into chilled 1l centrifuge pots on ice using a peristaltic pump. Harvest the cells by centrifugation at 3,500 x g, 4 &deg;C for 30 minutes in a large capacity rotor (e.g. 6 litre). Resuspend the cells in 150 ml ice cold CRB with protease inhibitors. From here on, all work should be carried out at 4 &deg;C.</li>
	<li>Lyse the cells by passing through a Constant Systems cell disrupter in 4 passes at 25, 30, 32 and 35 kPa, collecting the lysate on ice in each case. Alternatively, use a bead beater (Biospec) with an equal volume of acid-washed 0.5 mm glass beads and agitate for 3 minutes on full power. Transfer the lysate to 50ml Falcon tubes and pellet the cell debris by centrifugation at 3,500 x g, 4 &deg;C for 15 minutes in a benchtop centrifuge.</li>
	<li>Transfer the supernatant to chilled centrifuge tubes. Centrifuge at 14,000 x g, 4 &deg;C for 30 minutes in a centrifuge to remove mitochondria.</li>
	<li>Transfer supernatant to chilled ultracentrifuge tubes. Centrifuge at 200,000 x g, 4 &deg;C for 90 minutes in an ultracentrifuge to collect microsomes.</li>
	<li>Carefully decant and discard the supernatant and add 2ml ice cold CFTR buffer with protease inhibitors and 1mM DTT to each tube. Gently resuspend the pellets using a paintbrush, top up each tube with CFTR buffer and mix using a vortex mixer.</li>
	<li>Centrifuge the suspension at 200,000 x g, 4 &deg;C for 60 minutes in an ultracentrifuge, discard the supernatant and resuspend pellets in 2 ml ice cold CFTR buffer with protease inhibitors (no DTT) using a paintbrush.</li>
	<li>Pool the resuspended microsomes, adjust the final volume to 50 ml with CFTR buffer and mix well. Reserve a 1 ml aliquot for SDS-PAGE gel analysis, as described in 2.9 - 2.11. The microsomes can now be flash frozen in liquid nitrogen and then stored at -80 &deg;C until needed.</li>
	<li>CFTR can be extracted from microsomes using one of the following detergents: lithium perfluorooctonoate acid (LiPFO), tetradecanoly-lyso-phosphatidylglycerol (LPG14), n-dodecyl-&beta;-D- maltoside (DDM). Mix the microsomes with CFTR buffer, protease inhibitors and 5% detergent (w/w). If DDM is used, also add 300 mM NaCl to the buffer. Agitate at 4 &deg;C for 15 minutes on a tube rotator.</li>
	<li>Centrifuge the samples at 100,000 x g, 4 &deg;C for 1 hour in an ultracentrifuge. Retain the supernatant and take a small aliquot for SDS-PAGE gel analysis. CFTR may now be purified from the solublised material by immobilised metal affinity chromatography followed by size exclusion chromatography.</li>
</ol>
4. Representative Results
Transformation of yeast with the CFTR-containing plasmid is not 100% efficient. A representative small-scale screen of CFTR expression in selected colonies from a transformation experiment will yield about 1 in 4 colonies expressing the protein. A typical result from a screen of 5 colonies picked from a plate is shown in panel A of Fig. 3. One of the colonies shows a strong level of expression of the CFTR-GFP fusion protein which typically runs between the 250 kDa and 130 kDa markers, as shown. The CFTR-GFP fluorescence levels will vary considerably between experiments, with colony 4 in Fig. 3 showing at least 10x greater fluorescence than the intrinsic fluorescent band at about 70kDa. If expression levels of CFTR-GFP appear to give less fluorescence than the 70 kDa band, then it is probably worth re-transforming and choosing a colony with higher levels of CFTR-GFP expression. As shown in Fig. 3A, it is unlikely, even with a high expression level of CFTR-GFP, that the CFTR-GFP band will be discernable in the cell extract by Coomassie staining.
Once selected colonies have been grown in larger scale experiments, and microsomes isolated, the presence of CFTR-GFP within the microsomes will need to be assessed, as shown in Fig. 3B. The results of this experiment are important, not only to assess the efficiency of the induction of expression, but also to check that the microsomes have been prepared carefully and that proteolysis has been minimized. The results shown in Fig. 3B imply that in this experiment the CFTR-GFP expression is somewhat lower (as judged relative to the intrinsic 70kDa band) than in the small-scale experiment shown in panel A. However this impression is biased by the overexposure of the fluorescence detector in this measurement. This was because the experimenter was checking for the presence of proteolytic fragments of the CFTR construct. There is some evidence in this experiment for some fluorescent proteolytic fragments between the 130 kDa and 100 kDa markers, but these are very weak compared to the full-length CFTR-GFP band. With the protease inhibitors described here, we find little evidence for proteolytic degradation of CFTR after cell breakage. If significant proteolysis is observed, we recommend making fresh protease inhibitor stock solutions. We have also found that commercial protease inhibitor cocktail tablets are not as effective for this system. Growth of cells beyond 16 hr (post-induction) will give rise to decreased CFTR expression as shown in Figure 4. This is probably due to turnover of the protein, perhaps due to upregulation of the yeast protein quality control machinery6-9,13. It is therefore advisable to monitor CFTR expression levels after induction with galactose, if possible, as the optimal time to harvest the cells may vary from one laboratory to another.
<img alt="Figure 1" src="/files/ftp_upload/3860/3860fig1.jpg" /> 
Figure 1. The CFTR construct-containing yeast plasmid. The CFTR-GFP-8His fusion is inserted into the 2&mu; p424GAL1 expression vector, under the control of a galactose (GAL1) promoter. The vector also contains a uracil selection marker (URA) and an ampicillin resistance gene (Amp).
<img alt="Figure 2" src="/files/ftp_upload/3860/3860fig2.jpg" /> 
Figure 2. A flowchart summarising the visualised protocol. 
<img alt="Figure 3" src="/files/ftp_upload/3860/3860fig3.jpg" /> 
Figure 3. Representative SDS-PAGE gels of CFTR expression and purification. Panel A shows five randomly picked transformant colonies (lanes 1-5) that were screened for CFTR expression. Panel B shows microsomes that were isolated from a 15l fermenter culture. Panel C shows purified murine CFTR obtained after two-stage purification using affinity chromatography followed by size exclusion chromatography. All gels are shown under illumination conditions exciting fluorescence from the GFP domain (left) and after Coomassie stain (right). The relative locations of molecular weight standards are listed on the left (kDa).
<img alt="Figure 4" src="/files/ftp_upload/3860/3860fig4.jpg" /> 
Figure 4. Representative data for the expression of CFTR in yeast. Panel A shows a time-course of CFTR expression after induction with galactose. Cell extracts were analysed by SDS-PAGE, and the GFP fluorescence for the CFTR-GFP protein band was integrated. Panel B shows typical results for fluorescence microscopy of GFP-expressing cells 16 hr post induction. Typically, only a fraction of the cells express CFTR at high levels.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidylglycerol:+a+novel+effective+detergent+for+solubilizing+and+purifying+the+cystic+fibrosis+transmembrane+conductance+regulator.">Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.</a> Anal. Biochem. 259, 89-97 (1998).</li></ol>

No conflicts of interest declared.

This paper provides a method for the expression of murine CFTR protein in yeast cells, which should facilitate research on cystic fibrosis. The aim is to link this paper with the release of the murine CFTR DNA construct, which will be available through the Cystic Fibrosis Foundation (<a href="http://www.cff.org/research/CFFT/">http://www.cff.org/research/CFFT/</a>). Other orthologs should become available later. Transformation of the yeast cells with the CFTR-containing vector is straightforward, but it is important to screen for colonies expressing high levels of CFTR. Variable expression levels may arise from several factors, but the number of copies of the plasmid per cell probably accounts for a significant degree of variation. Critical steps described here should allow production of CFTR-expressing yeast cells and CFTR-containing microsomal membranes. Once the transformation, growth, harvesting and lysis of yeast cells have been mastered, purification of the protein should be possible, and in Figure 3 we have given an example of the purity that should be achievable in this case as a useful benchmark. It is not our intention in this manuscript to provide detailed methodology for purification of the protein. However, there are some critical downstream purification steps that are specific to the S.cerevisae expression system, such as cell lysis and microsome purification, and these have been included in detail in this manuscript. It should be mentioned, however, that apart from the two methods we have used, alternative yeast cell disruption methods can be employed, such as the use of a French pressure cell. The recombinant protein has a TEV-cleavable C-terminal GFP domain that allows the protein to be tracked after induction (Fig. 4). Yeast have an intrinsic 70kDa protein (probably succinate dehydrogenase12) that fluoresces under the same conditions11, and this can provide a useful internal calibration standard for the relative expression levels of CFTR in whole cell extracts or microsomes (Fig. 3). It is clear from the data shown in Figure 4 that the timing of cell harvesting after induction with galactose is crucial. Yields of CFTR drop precipitously after about 16 hr of induction, so that there is barely any detectable CFTR in yeast cells after 24 hr of induction. 
The yield of purified protein is about 1-2mg CFTR protein per 15 litre fermenter culture. Recovery can be estimated as about 70% of the total CFTR-GFP protein up to the microsome stage, and about 25% recovery of purified protein. Characterisation of the S. cerevisiae-expressed CFTR is ongoing. As seen in Fig. 4, the protein's location in the cell can be monitored by fluorescence microscopy. Although much of the fluorescence is found around the periphery of the cell as expected10, some of the protein displays a punctate localization, either in, or just inside the plasma membrane which could be due to CFTR recycling through a late Golgi/endosomal pathway14 or perhaps a compartment downstream of the budding of transport vesicles from the ER4. Treatment with PNGaseF, an enzyme that deglycosylates proteins, showed minimal change in the migration of the CFTR protein band on SDS-PAGE, implying that it is unglycosylated, or has minimal glycosylation 15. Experiments on the phosphorylation state of the protein are underway. In some of the detergents tested so far, the purified protein displays ATPase activity (that is inhibited by a CFTR-specific inhibitor16) at rates that are similar to those previously published 2,15 . Measurement of CFTR channel activity will require reconstitution of the purified protein, which would imply a final purification step in a detergent that has a relatively high critical micelle concentration (cmc)17. Yeast microsomes containing CFTR can be solublised with several commonly employed detergents18, including detergents such as dodecyl maltoside2, which are generally considered to be 'mild'. However most high cmc detergents have proven to be inefficient for solubilsation, so far, suggesting that exchange into these detergents should be considered at a late stage in any purification scheme.

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue No</td>
 </tr>
 <tr>
 <td>FGY217 S.cerevisiae strain, with pep4 deletion 10 </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Yeast nitrogen base without amino acids</td>
 <td>Formedium</td>
 <td>CYN0410</td>
 </tr>
 <tr>
 <td>Complete supplement mixture without uracil</td>
 <td>Formedium</td>
 <td>DCS0169</td>
 </tr>
 <tr>
 <td>Bacteriological agar </td>
 <td>Sigma-Aldrich</td>
 <td>A5306</td>
 </tr>
 <tr>
 <td>D-galactose</td>
 <td>Fisher</td>
 <td>BP656-500 </td>
 </tr>
 <tr>
 <td>D-glucose</td>
 <td>Fisher</td>
 <td>D16-500</td>
 </tr>
 <tr>
 <td>Pepstatin A</td>
 <td>Sigma-Aldrich</td>
 <td>P4265</td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>Merck</td>
 <td>108975 </td>
 </tr>
 <tr>
 <td>Chymostatin </td>
 <td>Sigma-Aldrich</td>
 <td>C7268</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonylfluoride (PMSF) </td>
 <td>Sigma-Aldrich</td>
 <td>P7626</td>
 </tr>
 <tr>
 <td>Epoxysuccinyl-leucylamido-butane (E-64) </td>
 <td>Sigma-Aldrich</td>
 <td>E3132</td>
 </tr>
 <tr>
 <td>Aminoethylbenzenesulfonyl fluoride (AEBSF) </td>
 <td>Sigma-Aldrich</td>
 <td>A8456</td>
 </tr>
 <tr>
 <td>Benzamidine hydrochloride </td>
 <td>Sigma-Aldrich</td>
 <td>434760</td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO) </td>
 <td>Sigma-Aldrich</td>
 <td>D8418</td>
 </tr>
 <tr>
 <td>dithiothreitol (DTT)</td>
 <td>Sigma-Aldrich</td>
 <td>43815</td>
 </tr>
 <tr>
 <td>ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher </td>
 <td>BP120500 </td>
 </tr>
 <tr>
 <td>Tris-base</td>
 <td>Formedium</td>
 <td>TRIS01</td>
 </tr>
 <tr>
 <td>Tris-HCl</td>
 <td>Fisher</td>
 <td>P631 </td>
 </tr>
 <tr>
 <td>D-sorbitol </td>
 <td>Sigma-Aldrich</td>
 <td>S1876</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Fisher</td>
 <td>065017</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S6191</td>
 </tr>
 <tr>
 <td>n-dodecyl-&beta;-D-maltopyranoside</td>
 <td>Affymetrix</td>
 <td>D310S</td>
 </tr>
 <tr>
 <td>Bromophenol blue</td>
 <td>Sigma-Aldrich</td>
 <td>114391 </td>
 </tr>
 <tr>
 <td>PageRuler Plus prestained protein standards </td>
 <td>Fermentas</td>
 <td>SM1811 </td>
 </tr>
 <tr>
 <td>NuSep tris-gly 4-20% gradient gels</td>
 <td>NuSep</td>
 <td>NB10-420</td>
 </tr>
 <tr>
 <td>Instant Blue Coomassie Stain</td>
 <td>Novexin</td>
 <td>ISB1L </td>
 </tr>
 <tr>
 <td>Glass beads, acid washed</td>
 <td>Sigma</td>
 <td>G8772</td>
 </tr>
 <tr>
 <td>50ml Sterile Falcon Tubes</td>
 <td>Sarstedt</td>
 <td>62.547.254</td>
 </tr>
 <tr>
 <td>2ml Sterile screw-top vials</td>
 <td>Sarstedt</td>
 <td>72.694.005 </td>
 </tr>
 <tr>
 <td>250ml Sterile Erlenmeyer baffled flasks </td>
 <td>BD Biosciences</td>
 <td>355119 </td>
 </tr>
 <tr>
 <td>2l Sterile Erlenmeyer baffled flasks </td>
 <td>BD Biosciences</td>
 <td>355131 </td>
 </tr>
 <tr>
 <td>2ml microfuge tubes</td>
 <td>Sarstedt</td>
 <td>72.695 </td>
 </tr>
 <tr>
 <td>0.2&mu;M syringe filter </td>
 <td>Sartorius</td>
 <td>FC121</td>
 </tr>
 <tr>
 <td>ultracentrifuge tubes</td>
 <td>Beckman Coulter</td>
 <td>355618</td>
 </tr>
 <tr>
 <td>centrifuge tubes</td>
 <td>Beckman Coulter</td>
 <td>357000 </td>
 </tr>
 <tr>
 <td>1l centrifuge pots</td>
 <td>Beckman Coulter</td>
 <td>969329 </td>
 </tr>
 <tr>
 <td>Orbital shaking incubator with temperature control </td>
 <td>New Brunswick Scientific</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deltavision RT restoration microscope </td>
 <td>Applied Vision</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Benchtop centrifuge </td>
 <td>HERMLE</td>
 <td>Z300</td>
 </tr>
 <tr>
 <td>Benchtop microfuge </td>
 <td>Fisher</td>
 <td>13-100-511 </td>
 </tr>
 <tr>
 <td>Vortex mixer</td>
 <td>Star Labs</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Typhoon Trio Scanner</td>
 <td>GE Healthcare</td>
 <td>63-0055-87</td>
 </tr>
 <tr>
 <td>LAS3000 imaging system</td>
 <td>Fuji</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>20l fermenter vessel and control unit</td>
 <td>Applikon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Constant systems cell disrupter</td>
 <td>Constant systems</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Beadbeater cell disrupter</td>
 <td>BioSpec</td>
 <td>1107900</td>
 </tr>
 <tr>
 <td>Mini-beadbeater-16</td>
 <td>BioSpec</td>
 <td>607</td>
 </tr>
 <tr>
 <td>JA-17 rotor</td>
 <td>Beckman Coulter</td>
 <td>369691 </td>
 </tr>
 <tr>
 <td>Optima L-100 Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td>392050</td>
 </tr>
 <tr>
 <td>50.2Ti rotor</td>
 <td>Beckman Coulter</td>
 <td><a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901" target="_blank">337901</a> </td>
 </tr>
 </tbody>
</table>

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.

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

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.

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  ...

expression-purification-cystic-fibrosis-transmembrane-conductance

Research

JoVE Journal

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 ...

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 ...

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.

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;

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

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 ...