High-level cell-free expression and functional characterization of a novel aquaporin from Photobactetrium profundum SS9

•The bioactivity of aquaporin in E.coli was demonstrated by in-vivo expression of AqpSS9 from P. profundum SS9.•A high productivity of soluble AqpSS9 (571 mg/l) was achieved by using in-vitro E. coli cell-free expression system (D-CF mode).•High water permeability (Pf 310.7 ± 3.2 μm/s) was determined with AqpSS9 incorporated proteoliposome.•The construction of AqpSS9 embedded layer-by-layer (LbL) membrane with improved water permeability and salt rejection.

AqpSS9 is a novel aquaporin derived from the deep sea bacterium Photobactetrium profundum SS9 and has attracted many attentions in developing water filtration biomimetic membranes. Functional characterization of AqpSS9 was carried out first by its expression in E. coli MM1211 (aqpZ−). Results showed that it was similar to bacterial aquaporin Z (AqpZ) and functioned as a real aquaporin. In-vitro expression of AqpSS9 were systematically investigated using three different modes: precipitate-based cell-free (P-CF) mode, the detergent-based cell-free (D-CF) mode and lipid-based cell-free expression mode (L-CF). D-CF mode showed more superiority than P-CF and L-CF mode, and the highest expression level of 571 mg/l was achieved by adding 0.7% Brij-78. Then AqpSS9 was purified by affinity chromatograph and incorporated into DOPC liposomes. Osmotic water permeability values (Pf) of reconstituted AqpSS9 proteoliposomes was measured as 310.7 ± 3.2 μm/s, which was about 3.5 times of empty control liposomes and comparable to reported Aqps. The AqpSS9 embedded layer-by-layer (LbL) membrane was fabricated and tested, which showed enhanced water permeability and salt rejection in comparison with the control membrane. This work demonstrated the good performance of AqpSS9 as a water channel protein, which may become an alternative candidate for biomimetic membrane construction for water filtration.

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