The Cell-free Expression And Function Characterization Of AqpSS9

With high peameability and selectivity to water molecule, Aquaporins may have potential application value in water recycling and desalination. In this work, the E.coli cell-free expression and functional assay were conducted on AqpSS9 from deep sea bacteria Photobacterium profundum SS9 as well as AqpZ from E.coli. Also, the Aq-uaproins embedded layer-by-layer (LbL) membrane was fabricated to demonstrate the function of Aquaroins.Firstly, the E.coli cell-free expression system was estabilished and the vector pl-VEX2.4c-AqpSS9 was constructed. In order to obtain soluble AqpSS9, three different cell-free expression modes (P-CF, D-CF and L-CF) were used.D-CF turned to be the best mode and the concentration of Brij-78 was screened. With the presence of 0.7% Brij-78, highest expression level of AqpSS9 was achieved which was 565 μg/mL. The soluble AqpSS9 was purified by Ni2+-NTA affinity chro-matograph and then reconstituted into DOPC liposomes whose reconstitution effiency was 55%. The water channel activity of AqpSS9 proteoliposomes was analyzed by stopped-flow. Osmotic water permeability value (Pf) of AqpSS9 proteoliposomes was 310.7 ± 3.2 μm/s, which was about 3.5 times higher compared to the control (89.3 ± 1.8 μm/s). The water permeability per AqpSS9 monomer (Pf,mono) can be estimated based on the proteoliposome area coverage of AqpSS9 to be 2.25 × 10-20m3/s. The NaCl refection coefficient σ of AqpSS9 proteoliposomes and control were 0.83 and 0.79, respectively.Secondly, soluble AqpZ was expressed by adding 1.0% Brij-78 to E.coli cell-free expression system, and was lately purified by Ni2+-NTA affinity chromatograph.490 μg/mL AqpZ protein solution was obtained after concentration. Then AqpZ was re-constituted into DOPC liposomes and its efficiency was 57%. The Pf value of AqpZ proteoliposomes was 258.1 ± 8.9 μm/s, which was about 2.8 times higher compared to the control (92.3 ± 1.2 μm/s).Finally, a biomimetic nanofiltration membrane was fabricated by immobilizing an aquaproin-incroporated supported lipid bilayer on a layer-by-layer complex polyelec-trolyte membrane. The nanofiltration and forward osmosis tests were performed on the biomimetic membranes with AqpSS9 and AqpZ to evaluate water flux and NaCl rejection. Furthermore, commercial PAN substrate membranes were used and nanofil-tration tests were onducted on the biomimetic membranes with AqpSS9, AqpZ and AqpZ-Mutant. The Aquaporins embedded membranes demonstrated a higher water flux and NaCl rejection because of the presense of Aquaporins.In conclution, soluble AqpSS9 and AqpZ were expressed in cell-free expression systm and function assay was conducted by stopped-flow experiments. Also, the aq-uaporins embedded membranes were fabricated. This work could be the foundation for the further study of Aquaporins.