Faculty of Life Sciences, University of Manchester
O'Ryan, L., Rimington, T., Cant, N., Ford, R. C. Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae. J. Vis. Exp. (61), e3860, doi:10.3791/3860 (2012).
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of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished).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 >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.
1. Preparation of Media and Buffers
|Inhibitor||Stock Concentration||Stock Preparation||Working Concentration|
|AEBSF||200 mM||Dissolve 48mg in 1ml distilled water||0.2 mM|
|Benzamidine||300 mM||Dissolve 36mg in 1ml distilled water||0.3 mM|
|Chymostatin||4 mM||Dissolve 2.5mg in 1ml dry DMSO||4 μM|
|E-64||7 mM||Dissolve 2.5mg in 1ml distilled water||7 μM|
|Leupeptin||20 mM||Dissolve 10mg in 1ml distilled water||20 μM|
|Pepstatin A||15 mM||Dissolve 10mg in 1ml dry DMSO||15 μM|
|PMSF||1 M||Dissolve 174mg in 1ml dry DMSO||1 mM|
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 °C. For longer term storage, glycerol stocks should be made and stored at -80 °C. Methods for cloning and transformation are described in detail by Drew et al. (2008)10.
3. Large-scale Fermenter Culture
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.
Figure 1. The CFTR construct-containing yeast plasmid. The CFTR-GFP-8His fusion is inserted into the 2μ 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).
Figure 2. A flowchart summarising the visualised protocol.
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).
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.
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 (http://www.cff.org/research/CFFT/). 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.
No conflicts of interest declared.
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.
|FGY217 S.cerevisiae strain, with pep4 deletion 10|
|Yeast nitrogen base without amino acids||Formedium||CYN0410|
|Complete supplement mixture without uracil||Formedium||DCS0169|
|Aminoethylbenzenesulfonyl fluoride (AEBSF)||Sigma-Aldrich||A8456|
|ethylenediaminetetraacetic acid (EDTA)||Fisher||BP120500|
|PageRuler Plus prestained protein standards||Fermentas||SM1811|
|NuSep tris-gly 4-20% gradient gels||NuSep||NB10-420|
|Instant Blue Coomassie Stain||Novexin||ISB1L|
|Glass beads, acid washed||Sigma||G8772|
|50ml Sterile Falcon Tubes||Sarstedt||62.547.254|
|2ml Sterile screw-top vials||Sarstedt||72.694.005|
|250ml Sterile Erlenmeyer baffled flasks||BD Biosciences||355119|
|2l Sterile Erlenmeyer baffled flasks||BD Biosciences||355131|
|2ml microfuge tubes||Sarstedt||72.695|
|0.2μM syringe filter||Sartorius||FC121|
|ultracentrifuge tubes||Beckman Coulter||355618|
|centrifuge tubes||Beckman Coulter||357000|
|1l centrifuge pots||Beckman Coulter||969329|
|Orbital shaking incubator with temperature control||New Brunswick Scientific|
|Deltavision RT restoration microscope||Applied Vision|
|Vortex mixer||Star Labs|
|Typhoon Trio Scanner||GE Healthcare||63-0055-87|
|LAS3000 imaging system||Fuji|
|20l fermenter vessel and control unit||Applikon|
|Constant systems cell disrupter||Constant systems|
|Beadbeater cell disrupter||BioSpec||1107900|
|JA-17 rotor||Beckman Coulter||369691|
|Optima L-100 Ultracentrifuge||Beckman Coulter||392050|
|50.2Ti rotor||Beckman Coulter||337901|