Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System
Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from <i>Escherichi...
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MDPI AG
2019-10-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/8/11/1325 |
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| author | Ke Yue Tran Nam Trung Yiyong Zhu Ralf Kaldenhoff Lei Kai |
| author_facet | Ke Yue Tran Nam Trung Yiyong Zhu Ralf Kaldenhoff Lei Kai |
| author_sort | Ke Yue |
| collection | DOAJ |
| description | Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from <i>Escherichia coli</i> and <i>Nicotiana tabacum</i> using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and <i>Nt</i>PIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms. |
| first_indexed | 2024-03-12T11:10:26Z |
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| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T11:10:26Z |
| publishDate | 2019-10-01 |
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| spelling | doaj.art-3c7c16732dc6404db850cb0235a2ffb82023-09-02T02:57:42ZengMDPI AGCells2073-44092019-10-01811132510.3390/cells8111325cells8111325Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis SystemKe Yue0Tran Nam Trung1Yiyong Zhu2Ralf Kaldenhoff3Lei Kai4The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou 22116, ChinaDepartment of Biology, Applied Plant Sciences, Technische Universität Darmstadt, Schnittspahn Strasse 10, D-64287 Darmstadt, GermanyJiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing 210095, ChinaDepartment of Biology, Applied Plant Sciences, Technische Universität Darmstadt, Schnittspahn Strasse 10, D-64287 Darmstadt, GermanyThe Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, School of Life Sciences, Jiangsu Normal University, Xuzhou 22116, ChinaAquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from <i>Escherichia coli</i> and <i>Nicotiana tabacum</i> using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and <i>Nt</i>PIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.https://www.mdpi.com/2073-4409/8/11/1325aquaporincell-free protein synthesisco-translational insertionproteo-liposome |
| spellingShingle | Ke Yue Tran Nam Trung Yiyong Zhu Ralf Kaldenhoff Lei Kai Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System Cells aquaporin cell-free protein synthesis co-translational insertion proteo-liposome |
| title | Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System |
| title_full | Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System |
| title_fullStr | Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System |
| title_full_unstemmed | Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System |
| title_short | Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System |
| title_sort | co translational insertion of aquaporins into liposome for functional analysis via an i e coli i based cell free protein synthesis system |
| topic | aquaporin cell-free protein synthesis co-translational insertion proteo-liposome |
| url | https://www.mdpi.com/2073-4409/8/11/1325 |
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