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Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System.

ABSTRACT: 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 Escherichia coli and Nicotiana tabacum 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 NtPIP2;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.

SUBMITTER: Yue K 

PROVIDER: S-EPMC6912355 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an <i>E. coli</i> Based Cell-Free Protein Synthesis System.

Yue Ke K   Trung Tran Nam TN   Zhu Yiyong Y   Kaldenhoff Ralf R   Kai Lei L  

Cells 20191027 11

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 wa  ...[more]

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