title: Preparative scale production and functional reconstitution of a human aquaglyceroporin (AQP3) using a cell free expression system.
abstract: Understanding the selectivity of aquaporin (AQP) membrane channels and exploiting their biotechnological potential will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of high quality membrane proteins. Here, we establish a convenient method to synthesize large amounts of functional human aquaglyceroporin 3 protein (AQP3), an AQP of physiological relevance conducting glycerol and some small neutral solutes besides water. Milligram amounts of AQP3 were produced as a histidine-tagged protein (hAQP3-6His) in an Escherichia coli extract-based CF system in the presence of the non-ionic detergent Brij-98. The recombinant AQP3 was purified by affinity chromatography, incorporated into liposomes and evaluated functionally by stopped-flow light scattering. Correct protein folding was indicated by the high glycerol and water permeability exhibited by the hAQP3-6His proteoliposomes as compared to empty control liposomes. Functionality of hAQP3-6His was further confirmed by the strong inhibition of the glycerol and water permeability by phloretin and HgCl2, respectively, two blockers of AQP3. Fast and convenient CF production of functional AQP3 may serve as basis for further structural/functional assessment of aquaglyceroporins and help boosting the AQP-based biomimetic technologies.
authors.affiliation: Department of Pharmaceutical and Medical Chemistry, University of Kiel, Kiel, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Müller-Lucks
authors.forename: Patrizia
authors.initials: P
authors.lastname: Gena
authors.forename: Daniele
authors.initials: D
authors.lastname: Frascaria
authors.forename: Nicola
authors.initials: N
authors.lastname: Altamura
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.forename: Eric
authors.initials: E
authors.lastname: Beitz
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Calamita
mesh_terms: D051400:Aquaporin 3 / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008627:Mercuric Chloride / Q000737:chemistry; D010693:Phloretin / Q000737:chemistry; D010938:Plant Oils / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: C496027:AQP3 protein, human; D008081:Liposomes; D010938:Plant Oils; D011993:Recombinant Fusion Proteins; D051400:Aquaporin 3; D011092:Polyethylene Glycols; D008627:Mercuric Chloride; C033084:polyethylene glycol oleyl ether; D005990:Glycerol; D010693:Phloretin