Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this ‘difficult’ eukaryotic membrane protein.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in 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of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished).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.
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.
The authors have nothing to disclose.
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.
Name of reagent/equipment | Company | Catalogue No |
FGY217 S.cerevisiae strain, with pep4 deletion 10 | ||
Yeast nitrogen base without amino acids | Formedium | CYN0410 |
Complete supplement mixture without uracil | Formedium | DCS0169 |
Bacteriological agar | Sigma-Aldrich | A5306 |
D-galactose | Fisher | BP656-500 |
D-glucose | Fisher | D16-500 |
Pepstatin A | Sigma-Aldrich | P4265 |
Leupeptin | Merck | 108975 |
Chymostatin | Sigma-Aldrich | C7268 |
Phenylmethanesulfonylfluoride (PMSF) | Sigma-Aldrich | P7626 |
Epoxysuccinyl-leucylamido-butane (E-64) | Sigma-Aldrich | E3132 |
Aminoethylbenzenesulfonyl fluoride (AEBSF) | Sigma-Aldrich | A8456 |
Benzamidine hydrochloride | Sigma-Aldrich | 434760 |
Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D8418 |
dithiothreitol (DTT) | Sigma-Aldrich | 43815 |
ethylenediaminetetraacetic acid (EDTA) | Fisher | BP120500 |
Tris-base | Formedium | TRIS01 |
Tris-HCl | Fisher | P631 |
D-sorbitol | Sigma-Aldrich | S1876 |
Glycerol | Fisher | 065017 |
NaCl | Sigma-Aldrich | S6191 |
n-dodecyl-β-D-maltopyranoside | Affymetrix | D310S |
Bromophenol blue | Sigma-Aldrich | 114391 |
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 | |
Benchtop centrifuge | HERMLE | Z300 |
Benchtop microfuge | Fisher | 13-100-511 |
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 |
Mini-beadbeater-16 | BioSpec | 607 |
JA-17 rotor | Beckman Coulter | 369691 |
Optima L-100 Ultracentrifuge | Beckman Coulter | 392050 |
50.2Ti rotor | Beckman Coulter | 337901 |