Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.
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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3569440?pdf=render |
| _version_ | 1819041894984843264 |
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| author | Julie Bomholt Claus Hélix-Nielsen Peter Scharff-Poulsen Per Amstrup Pedersen |
| author_facet | Julie Bomholt Claus Hélix-Nielsen Peter Scharff-Poulsen Per Amstrup Pedersen |
| author_sort | Julie Bomholt |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T09:32:15Z |
| format | Article |
| id | doaj.art-0fe13450eff1420480d8998ce7a4304b |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T09:32:15Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0fe13450eff1420480d8998ce7a4304b2022-12-21T19:08:43ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5643110.1371/journal.pone.0056431Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.Julie BomholtClaus Hélix-NielsenPeter Scharff-PoulsenPer Amstrup PedersenIn 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.http://europepmc.org/articles/PMC3569440?pdf=render |
| spellingShingle | Julie Bomholt Claus Hélix-Nielsen Peter Scharff-Poulsen Per Amstrup Pedersen Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. PLoS ONE |
| title | Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. |
| title_full | Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. |
| title_fullStr | Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. |
| title_full_unstemmed | Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. |
| title_short | Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae. |
| title_sort | recombinant production of human aquaporin 1 to an exceptional high membrane density in saccharomyces cerevisiae |
| url | http://europepmc.org/articles/PMC3569440?pdf=render |
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