title: Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.
abstract: In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.
authors: Aquaporin A/S, Copenhagen, Denmark.
authors: Julie
authors: J
authors: Bomholt
authors: Claus
authors: C
authors: Hélix-Nielsen
authors: Peter
authors: P
authors: Scharff-Poulsen
authors: Per Amstrup
authors: PA
authors: Pedersen
mesh_terms: D051398:Aquaporin 1 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D005818:Genetic Engineering; D005960:Glucosides / Q000737:chemistry; D006801:Humans; D009532:Nickel / Q000737:chemistry; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics*; D012995:Solubility; D013696:Temperature; D013997:Time Factors
chemical_list: D003902:Detergents; D005960:Glucosides; D011994:Recombinant Proteins; C400643:cyclohexyl-pentyl-maltoside; D051398:Aquaporin 1; D009532:Nickel