(191p) Improved Performance of Biomimetic Membrane Integrated with the Aquaporins Modified with in-Vitro Genetic Incorporation of P-propargyloxyphenylalanine

Aquaporins (AQPS) are widely applied in biomimetic membranes for water recycling and desalination. To improve the stability of AQPS in biomimetic membranes, modified AQP Z and AQP SS9 were obtained by incorporation of an unnatural amino acid (p-propargyloxyphenylalanine, pPpa) into AQPS in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) via the cell free expression system. The modified AQPS can realize the covalent-link with phospholipids in membrane and hence improve the stability of biomimetic membrane greatly. The concentration of Mg²⁺ and fusion expression with signal peptides were evaluated to enhance the expression level of modified AQP SS9 and AQP Z. The modified AQP SS9 and AQP Z were then reconstituted into DOPC liposome and analyzed by stopped-flow spectrophotometer. The obtained water permeability coefficient (P_f) of 7.46x10^-4 m/s was 5.7 times higher than that of proteoliposomes with the original AQP SS9 (Pf=1.31x10^-4 m/s), and 6.8 times higher than that of the DOPC liposomes (Pf=5.01x10^-5m/s). The membrane embedded with modified AQP SS9 has not only showed improved stability, but also exhibited a higher water flux and NaCl rejection, than those of natural AQP SS9. Similar results were also obtained with modified AQP Z. The research was helpful to develop biomimetic membranes with highly stable aquaporins.