title: Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae.
abstract: 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 protein(1-3). 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 described(6-9) .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of the CFTR construct employed in  this report will be published separately (Urbatsch, I.; Thibodeau, P. et al.,  unpublished).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.
authors.affiliation: Faculty of Life Sciences, University of Manchester.
authors.forename: Liam
authors.initials: L
authors.lastname: O'Ryan
authors.forename: Tracy
authors.initials: T
authors.lastname: Rimington
authors.forename: Natasha
authors.initials: N
authors.lastname: Cant
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine