Name: efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format
Uploaded: 2016-09-17T14:00:00
Description: Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

This work was supported by a Joseph P. Healey grant from the University of Massachusetts Boston (L.S.H.) and R15 GM86805 from the NIH (L.S.H.). S.M.P. is supported in part by a Sanofi-Genzyme Fellowship at the University of Massachusetts Boston.

1. Preparing for Sporulation
Note: The media described in this protocol are made using standard recipes and methods13,17. Table 1 gives the formulation for 1 L of the various media used in this protocol.
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<ol>
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Here we present a protocol for sporulating SK1 yeast in a 96 multiwell format. Aeration is key for efficient sporulation, which requires the use of either a stir bar or a glass bead in each well. When cells are sporulated in a 96 multiwell plate in a shaking incubator without either a bead or a stir bar, cells do not sporulate efficiently. Only a small increase in sporulation efficiency is seen when cells are sporulated without either a bead or a stir bar in a shaking incubator, compared to being at 30 &#176;C without ag...

Sporulation in the budding yeast has been studied to provide insights into many aspects of biology, including the control of chromosome segregation during meiosis1, mechanisms of genetic recombination2, the control of development by cell signaling3, nutritional control of development4, the transcriptional regulation of development5, and the examination of spore formation6. Spore formation includes a novel cell division event involving the formation of new membrane compartments within the mother cell followed by the deposition of a protective spore wall6. These studies that examine sporulating cells often take advantage of the rapidly sporulating yeast strain SK1, which can undergo the process of sporulation in about 24 hr in a relatively efficient fashion7,8. Although optimization of sporulation conditions for budding yeast have been described9-13, these experiments examined sporulation on solid media or in larger scale liquid cultures where sporulation is carried out using culture tubes or flasks.
Here we describe a method for sporulating yeast in a 96 multiwell plate format. We find that for this method, aeration is critical for synchronous and efficient sporulation, and have devised techniques to ensure sufficient sporulation a small-volume multiwell format. Sporulating in a 96 multiwell plate format allows for cells to be screened using high-throughput techniques and reagents optimized for a multiwell plate format, such screening for high copy suppressors using a tiled library14-16.

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
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			<td height="21" style="height:21px;width:216px;">Nunc 1.3 ml DeepWell Plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">260251</td>
			<td style="width:288px;">Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nunc 2.0 ml DeepWell plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td>278743</td>
			<td>Used for presporulation growth, step 1.2.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mm glass bead</td>
			<td style="width:123px;">Fisher</td>
			<td>11-312A</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 mm x 2 mm stir bar, pack of 12</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">14-511-82</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96 well frogger</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP407</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">library copier</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP381</td>
			<td>needed for step 1.2; to be used with the frogger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rectangular petri dish</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">264728</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Peptone</td>
			<td style="width:123px;">BD</td>
			<td>211677</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:123px;">BD</td>
			<td>212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">D16-3</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Acetate</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">P171-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">G33-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Black 96 well glass bottom plate</td>
			<td style="width:123px;">MatTek</td>
			<td style="width:119px;">PBK96G-1.5.5-F</td>
			<td>needed for step 2.4</td>
		</tr>
	</tbody>
</table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

To assess this protocol, sporulation efficiencies obtained from sporulating cells in multiwell plates (as described above) were compared to cells sporulated using larger volumes in flasks (Table 2). The use of multiwell plates did not achieve the high efficiency seen when sporulating in flasks, where ~80% efficiency can be routinely seen. Sporulating in multiwell plates with proper aeration (provided by glass beads or stir bars) can achieve sufficient sporulation efficien...

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

The overall goal of this protocol is to sporulate budding yeast efficiently in a 96-well format. Although many large-scale screens have been carried out on the budding yeast, high-throughput studies looking at meiosis and sporulation have been limited. The technique described in this video has the advantage of sporulating yeast efficiently in a cost-effective manner in a 96-well format compatible with high-throughput screening.I first got the idea for this method because I wanted to use a tiled two micron plasmid library in order to screen for high copy suppressors of a mutant that could not form refractile spores. Initially, my sporulation efficiencies were poor in the 96-well format. I found this was due to inadequate aeration.Begin this procedure by thawing the SK1 yeast strain onto a yeast extract peptone glycerol or YPG plate. Use a sterile stick to scrape a small amount of cells out of the freezer vial containing the glycerol stock and spread it onto a YPG plate. Grow the yeast for 12 to 16 hours at 30 degrees Celsius.Next, streak the yeast from the YPG plate onto a yeast extract peptone dextrose, or YPD, plate for single colones. Grow the yeast on the YPD plate for 24 to 48 hours at 30 degrees Celsius. After 24 to 48 hours of growth on YPD solid media, start a 25 milliliter YPD culture using a single colony from the YPD plate.Grow this culture overnight in a 30 degree Celsius shaking incubator at 220 RPM until the cells reach an OD600 of 4.0 to 7.0. On the following day, use an appropriate amount of cells from the YPD culture to start a 125 milliliter culture in yeast extract peptone acetate, or YPA, with an OD600 of 0.1. Grow the culture in a 30 degree Celsius shaking incubator at 220 RPM until the OD600 is between 1.3 and 1.5.Transfer the culture to centrifuge tubes and spin at 1800X G in a tabletop centrifuge for five minutes to pellet the cells. Wash the cells once with a 0.5 volume of sterile autoclaved deionized water. Spin again and discard the water.Resuspend the cells in a 1X volume of sporulation media. To prepare cells of different genotypes, cells from thawed frozen stocks in a 96 multiwell plate are first transferred onto YPG solid media. Flame a 96 prong frogger and then cool it momentarily on a plate of solid media before touching it to the yeast colonies.Grow the yeast overnight at 30 degrees Celsius. On the following day, transfer the cells from YPG solid media onto the YPD or selective solid media, depending on the genotype of the strain. YPD is used in this demonstration.Grow the yeast at 30 degrees Celsius until colonies are formed for each strain. Aliquot 1.2 milliliters of YPD liquid media into each well of a 96 deep well plate. Transfer the cells from the solid media into liquid media.Cover the plate with a plastic lid from a rectangular petri plate and hold the lid in place using a piece of tape to minimally restrict air circulation. Grow the cells overnight in a 30 degree Celsius shaker at 220 RPM. On the following day, centrifuge at 1800X G for five minutes.Pour off the media and add 500 microliters of YPA to each well. And resuspend the pelleted cells. Cover the plate with a plastic lid held in place by tape.Grow the cells for 15 hours in a 30 degree Celsius shaking incubator at 220 RPM. To provide proper aeration for efficient sporulation, add into each well of a 96-well plate either one three millimeter sterile solid glass bead or a sterile five millimeter by two millimeter magnetic stir bar. If all cells are of the same genotype, aliquat 500 microliters of cells plus media into each well of the 96-multiwell plate.Cover the plate with a plastic lid held in place by tape. If cells are of different genotypes, spin the cells that are in YPA at 1800X G for five minutes and pour off the media. Add 500 microliters of sporulation media to each well.And resuspend the pelleted cells in the sporulation media. Cover the plate with a plastic lid held in place by tape. If using glass beads, place the samples in a shaking incubator at 30 degrees Celsius and shake at 220 RPM for sporulation.If using magnetic stir bars, place the samples on a stir plate inside of a 30 degrees Celsius incubator for sporulation. The exact stir speed for the magnetic bars is not very important, as long as all of the bars are moving energetically and the culture is aerated. 36 hours later, retrieve the samples to monitor progression through sporulation.Withdraw six microliters of cells from each sporulating culture and add to 24 microliters of sporulation media in a 96-well coverslip plate. Leave the cells to settle for five minutes before examining them using an inverted microscope. A representative sporulation of two different strains is shown in these images of cells visualized using a 63X objective on an inverted microscope.Refractile tetrads, indicated by the arrowheads, can be seen in the wild-type culture. But not in the culture containing the sporulation-deficient smk1 delete cells. Comparisons were made between sporulation efficiencies from cells sporulated in multi-well plates and cells sporulated using larger volumes in flasks.The use of multi-well plates did not achieve the high efficiency routinely seen when using the more typical protocol of sporulating in flasks. Sporulating in multi-well plates with proper aeration can achieve sufficient sporulation efficiencies greater than 50%with the best results obtained using a five millimeter stir bar. However, given the high cost of the small stir bars, the efficiencies obtained using glass beads are comparable.Although both five millimeter and seven millimeter stir bars fit within the well of a 96-multiwell plate, using a seven millimeter stir bar resulted in poor sporulation efficiency. The seven millimeter stir bar in a well tends to interact with a stir bar in an adjacent well. Preventing the bars from stirring properly and providing adequate aeration to the culture.After watching this video, you should have a good understanding of how to efficiently sporulate yeast cells in liquid culture in the 96-well format. Once mastered, this process can be completed in six days, if done properly. This procedure can be used for screening different mutant strains, plasmids, or small molecule inhibitors for their effect on meiosis and sporulation.After it's development, this technique was used to study the regulation of meiotic cell cycle and how cytokinesis and meiosis are coupled in sporulation in Saccharomyces cerevisiae.

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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 ...

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 ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...