title: Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae.
abstract: BACKGROUND
  Decades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms.
  
  
  RESULTS
  During this work, a novel promoter system based on the nitrogen catabolite regulation has been developed to produce the general amino acid permease (Gap1) in its natural host, the yeast Saccharomyces cerevisiae. A simple purification protocol was also established that allows to purify milligrams of Gap1 from cells cultivated in a five liters bio-reactor. In order to test the ability of the system to be used for expression of other proteins, the yeast specific transporter of γ-aminobutyric acid (Uga4), a human vesicular transporter of glutamate (Vglut1) and a small secreted glycoprotein (MD-2) were also expressed using the nitrogen catabolite regulation. All proteins were fused to GFP and their presence and localization were confirmed by western blot analysis and fluorescence microscopy.
  
  
  CONCLUSIONS
  Our work shows that the nitrogen catabolite repressible GAP1 promoter can be used to obtain high levels of recombinant protein while allowing for large biomass production in S. cerevisiae. This approach can be used to express membrane and soluble proteins from higher eukaryotes (from yeast to human). Therefore, this system stands as a promising alternative to commonly used expression procedure in yeasts.
authors.forename: Fabien
authors.initials: F
authors.lastname: Debailleul
authors.forename: Cataldo
authors.initials: C
authors.lastname: Trubbia
authors.forename: Nancy
authors.initials: N
authors.lastname: Frederickx
authors.forename: Elsa
authors.initials: E
authors.lastname: Lauwers
authors.forename: Ahmad
authors.initials: A
authors.lastname: Merhi
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Ruysschaert
authors.forename: Bruno
authors.initials: B
authors.lastname: André
authors.affiliation: S,F,M,B,, Université Libre de Bruxelles, Blvd, du Triomphe, Bâtiment BC, local 1C4,208, B-1050 Bruxelles, Belgium. Cedric.Govaerts@ulb.ac.be.
authors.forename: Cédric
authors.initials: C
authors.lastname: Govaerts
mesh_terms: D000595:Amino Acid Sequence; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D009584:Nitrogen