Preparation And Characterization Of P-propargyloxyphenylalanine Incorporated Aquaporins

With high peameability and selectivity to water molecule, Aquaporins has been widely applied in biomimetic membranes for water recycling and desalination, But generally have the same weakness in membrane strength and stability. Change in the combination mode between Aquaporins and membrane material is the key to improve the membrane strength and stability of aquaporins based biomimetic membranes.In this research,by mutating the tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii, we had selectively and site-specifically incorporated the unnatural amino acids p-propargyloxyphenylalanine (pPpa) into AquaporinZ (AQPZ) and AquaporinSS9 (AQPSS9) respectively, and characterized its function, providing AQPs-based biomimic membranes with protein.Cell free (CF) expression system has been employed as a promising method to incorporate pPpa into AQPZ and AQPSS9. The concentration of Mg2+ was screened, resulting in a highest AQPZ yield of 48 mg/L,and fusion of leader peptides were investigated to enhance the expression of AQPSS9, resulting in a highest AQPSS9 yield of 49 mg/L. AQPs were purified by Ni2+ loaded NTA resin slurry.The modified AQPZ and AQPSS9 ware then reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes and analyzed by stopped-flow. The result showed that Osmotic water permeability value (Pf) of modified AQPZ proteoliposomes was 3.42x10-4 m/s,2.7 times higher compared to the AQPZ proteoliposomes(1.23×10-4 m/s) and 6.8 times higher compared to the DOPC liposomes (Pf=5.01×l0-5m/s), The Osmotic water permeability valueof modified AQPSS9 proteoliposomes was 7.46×10-4 m/s,5.7 times higher compared to the AQPSS9 proteoliposomes (Pf=1.31×10-4 m/s) and 12.1 times higher compared to the DOPC liposomes (Pf=5.01×l0-5m/s). It demonstrates that the incorporation of pPpa has no influence on high peameability of both AQPZ and AQPSS9.The modified AQPZ embedded layer-by-layer membrane demonstrated a higher water flux (19.9 L·m-2·h-1) and NaCl rejection (68.3%) compared to the AQPZ embedded membrane(water flux:18.7 L·m-2·h-1; NaCl rejection:60.7%). The modified AQPZ embedded membrane shows an equal water flux (14.8L·m-2h-1) and NaCl rejection (48.3%) to AQPZ embedded membrane.The research plays an important role in development of high stability aquaporins based biomimetic membranes.