Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › fagfællebedømt
Dokumenter
- Recombinant...
Forlagets udgivne version, 577 KB, PDF-dokument
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.
Originalsprog | Engelsk |
---|---|
Artikelnummer | e56431 |
Tidsskrift | P L o S One |
Vol/bind | 8 |
Udgave nummer | 2 |
Antal sider | 9 |
ISSN | 1932-6203 |
DOI | |
Status | Udgivet - 2013 |
Bibliografisk note
Artikel ID: e56431
Antal downloads er baseret på statistik fra Google Scholar og www.ku.dk
Ingen data tilgængelig
ID: 44741505