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.

Keywords: aquaporin; cell-free protein synthesis; co-translational insertion; proteo-liposome.

Figure 1

Screening of liposome concentration for CF expression of AqpZ-sGFP. The final concentrations of lipids were indicated with numbers below the bar chart. The reaction mixture with 8.6 mg/mL final lipid concentration without the AqpZ-sGFP plasmid template was used as the negative control. D-CF sample was used as positive control. RFU, relative fluorescent unit.

Figure 2

Confocal microscopy analysis of AqpZ-sGFP pellet fractions produced from l-CFPS directly after expression. (A,D), signals of sGFP in the pellet fractions shown in green; (B,E), signals of lipids in the pellet fractions in red (stained via Nile red); (C,F), merged images of the green and red channels. Scale bar indicates 10 μm.

Figure 3

Water permeability of AqpZ-sGFP and NtPIP2;1 proteo-liposome reformed via a detergent re-solubilization of the L-CFPS produced sample. (A,B), normalized and averaged light scattering measurements of AqpZ-sGFP and NtPIP2;1 proteo-liposome under osmotic shock via a stopped-flow spectrophotometry (n = 8–10). Red trace, proteo-liposome formed via the step dialysis method; blue trace, proteo-liposome formed via bio-beads; black trace, control empty liposomes. (C), bar chart of the calculated water permeability P_f (mean ± SD) of corresponding samples.

Figure 4

Monitoring the insertion of AqpZ-sGFP after reforming of the proteo-liposome. sGFP and lipids (stained via Nile red) are shown in green and red; a merged image is shown in C and F. (A–C), representative sample of the AqpZ-sGFP proteo-liposome formed after removal of detergents either via dialysis or bio-beads. The white arrows indicate the position of co-localized signals. (D–F), GUVs formed via fusion of AqpZ-sGFP proteo-liposome.