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在酿酒酵母中以极高的膜密度重组生产人水通道蛋白-1。

Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

机构信息

Aquaporin A/S, Copenhagen, Denmark.

出版信息

PLoS One. 2013;8(2):e56431. doi: 10.1371/journal.pone.0056431. Epub 2013 Feb 11.

DOI:10.1371/journal.pone.0056431
PMID:23409185
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3569440/
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.

摘要

在本研究中，我们探索了酿酒酵母（Saccharomyces cerevisiae）作为异源表达人水通道蛋白-1（Aquaporin-1）的宿主的能力。Aquaporin-1 cDNA 由位于具有可调节拷贝数质粒上的半乳糖诱导启动子表达。人 Aquaporin-1 通过酵母增强型 GFP（yeast enhanced GFP）进行 C 端标记，用于定量功能性表达、确定亚细胞定位、估计体内折叠效率以及建立纯化方案。在酵母宿主中过量表达 Gal4p 转录激活因子并在氨基酸补充的最小培养基中生长，在 15°C 下表达时，Aquaporin-1 构成总膜蛋白含量的 8.5%。凝胶内荧光结合 Western blot 显示，30°C 时，正确折叠的重组 Aquaporin-1 的低积累是由于体内错误折叠所致。将表达温度降低到 15°C 几乎完全防止了 Aquaporin-1 的错误折叠。活酵母细胞的生物成像显示，重组 Aquaporin-1 积累在酵母质膜中。去污剂筛选用于溶解，结果表明 CYMAL-5 在溶解重组 Aquaporin-1 方面更具优势，并生成单分散蛋白制剂。使用单个 Ni 亲和层析步骤即可获得几乎纯的 Aquaporin-1。与在人红细胞中发现的蛋白质相比，在 S. cerevisiae 中产生的重组 Aquaporin-1 不进行 N-糖基化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/d8830067b211/pone.0056431.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/434e44f653df/pone.0056431.g001.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/63b97bd82424/pone.0056431.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/cdb200ffe8a8/pone.0056431.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/e6c125af345d/pone.0056431.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/e3d25672d8e9/pone.0056431.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/d8830067b211/pone.0056431.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/434e44f653df/pone.0056431.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/d6a73c3c570e/pone.0056431.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/2f94463aeeb8/pone.0056431.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/421b45494eae/pone.0056431.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/63b97bd82424/pone.0056431.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/cdb200ffe8a8/pone.0056431.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/e6c125af345d/pone.0056431.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/e3d25672d8e9/pone.0056431.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5fda/3569440/d8830067b211/pone.0056431.g009.jpg

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2
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3
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PLoS One. 2023 Feb 21;18(2):e0281868. doi: 10.1371/journal.pone.0281868. eCollection 2023.
4
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Int J Mol Sci. 2022 Feb 23;23(5):2458. doi: 10.3390/ijms23052458.
5
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6
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