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.

Functional Analysis of Aquaporin Water Permeability Using an Escherichia coli-Based Cell-Free Protein Synthesis System

“…The water transport experiments were performed at 10 • C. (A-C) mAQP4 M23 expressed as pellet fractions and resolubilized in different detergents; (D) mAQP4 M23 expressed using D-CFPS (CFPS in the presence of detergent); (E,F) Escherichia coli AqpZ-sfGFP and NtPIP2;1 (from Nicotiana tabacum) expressed using L-CFPS (CFPS in the presence of liposomes) and treated with different detergent removal methods to reform the proteoliposomes. Panels (A-D) were adapted and reused from Kai et al (2010), PloS ONE, under the terms of the Creative Commons Attribution License; (E,F) were adapted and reused from Yue et al (2019), Cells, under the terms of the Creative Commons Attribution License.…”

“…Aquaporins are integral membrane proteins that facilitate the rapid and selective movement of water and neutral low-molecular-mass solutes across biological membranes along osmotic gradients. Although the function and structure of aquaporins have been extensively characterized ( Preston et al, 1992 ; Agre et al, 1993 ; Verkman and Mitra, 2000 ; Tornroth-Horsefield et al, 2006 ), sample preparation for the functional analysis of aquaporins is still challenging and time-consuming ( Yue et al, 2019 ). Three main methods are commonly used to determine the water permeability of aquaporins: (i) The Xenopus laevis oocyte system.…”

“…The first two methods do not require isolation and purification of aquaporins; however, they suffer from the influence of endogenous integral membrane proteins ( Ho et al, 2009 ). Although the liposome system offers a precise measurement of the permeability of specific aquaporins, the sample preparation process is still laborious and challenging, especially when aquaporins are overexpressed in vivo ( Yue et al, 2019 ). In contrast, the Cell-free Protein Synthesis (CFPS) system, which is devoid of living cells and cell membrane barriers, has the unique advantage of being an open system ( Henrich et al, 2015 ; Rues et al, 2016 ), allowing the introduction of various additives in a co-translational manner ( Schwarz et al, 2007 , 2008 ).…”

“…During the last few years, we have developed a set of protocols for the production and functional characterization of aquaporins based on the CFPS system ( Kai et al, 2010 ; Kai and Kaldenhoff, 2014 ; Yue et al, 2019 ). Here, we first detail a number of detergents suitable for soluble aquaporin expression.…”

“…Secondly, we provide a detailed purification strategy, including detergent exchange on a column for the downstream reconstitution process. Furthermore, based on the work of Hovijitra et al (2009) , we describe a detailed protocol for the direct insertion of cell-free (CF) expressed aquaporins into liposomes using a modified procedure that is suitable for the continuous exchange cell-free (CECF) expression mode ( Kai and Kaldenhoff, 2014 ; Yue et al, 2019 ). In this study, we have summarized our previous methods and provided a systematic protocol for the expression and functional characterization of aquaporins based on an Escherichia coli CFPS system, including template design, aquaporin expression and purification, proteoliposome preparation, and an aquaporin water permeability assay using stopped-flow spectrophotometry.…”