Expression And Bioactivity Evaluation Of A Novel Aquaporin Gene From Photobacterium Profundum SS9

Aquaporin (AQP), the natural water channel protein, exhibits high permeability to water, which makes AQP-based water filter a promising tool for water reuse and seawater desalination. This work aims to express a putative aquaporin aqpSS9 gene from a deep-sea model barophilic bacterium Photobacterium profundum SS9 in different host system, and one bioacitivity evaluation was carried out afterwards.Firstly by bioinformation analysis, AqpSS9 is 67% identical with E. coli AqpZ in amino acid sequence, and contains 2 NPA motifs and six bilayer-spanning domains. Furthermore it shows strong hydrophobicity and is predicted to integrate in the cell membrane to exhibit high water permeability.AqpSS9 gene aqpSS9 was coloned by PCR from P. profundum SS9 genome to construct expression vectors pUC19-AqpSS9 and pSJ2-AqpSS9. The reconstituted E.coli strains MM/pUC19-AqpSS9 and MM/pSJ2-AqpSS9 were co-cultured with the AqpZ- strain MM1211(aqpZ-) in hypotonic M9 medium respectively. Given that aquaporin would remarkably facilitate rapid growth of E. coli in hypoosmotic environment, by observing the dramatic growing predominance (a ratio peak of 32) of each reconstituted strain to MM1211(aqpZ-), AqpSS9 was indicated to perform water-uptake like aquaporin.Then pSJ2-AqpSS9 was transformed into E. coli to express AqpSS9 in prokaryotic system. Three typical E. coli hosts were compared, and E. coli Rossetta(DE3) was determined to be the most efficient one. After systematically investigating different induction conditions, such as induction-timing, temperature, IPTG concentration and post-induction duration, AqpSS9 productivity reached 48 mg/L. To overcome cell toxicity by membrane protein overexpression, Bac-to-Bac/BmNPV Baculovirus expression system was employed as an alternative to synthesize AqpSS9. Shuttle vector Bacmid-AqpSS9 was constructed to transfect the cultured BmN cells, and then got packaged to form recombinant baculovirus rBmAqpSS9. After repeated amplification a baculoviral stock with a suitable titer was prepared and infected BmN cells using an MOI of 5 for AqpSS9 expression. A high productivity of AqpSS9 was achieved (130 mg/L).This work reveals a novel marine aquaporin AqpSS9 and succeeds to express it in both prokaryotic and eukaryotic systems, which lays a solid foundation for further exploration.