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  • Plant Physiol Biochem. 2014-12; NULL NULL NULL
  • Functional reconstitution of a rice aquaporin water channel, PIP1; 1, by a micro-batchwise methodology.

  • V Scalera, P Gena, M Mastrodonato, Y Kitagawa, S Carulli, M Svelto, G Calamita.
  • Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy.

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...Acrylamide gels were stained with Coomassie Brilliant Blue (Sigma, St Louis, MO) or submitted to immunoblotting using an anti-6His tag monoclonal antibody (GenScript Corporation, Piscataway, NJ) at a final concentration of 0.2 μg/mL blocking buffer according to a previous...


Plant Physiol Biochem.

Abstract

Assessing the selectivity, regulation and physiological relevance of aquaporin membrane channels (AQPs) requires structural and functional studies of wild type and modified proteins. In particular, when characterizing their transport properties, reconstitution in isolation from native cellular or membrane processes is of pivotal importance. Here, we describe rapid and efficient incorporation of OsPIP1;1, a rice AQP, in liposomes at analytical scale. PIP1;1 was produced as a histidine-tagged form, 10His-OsPIP1;1, in an Escherichia coli-based expression system. The recombinant protein was purified by affinity chromatography and incorporated into liposomes by a micro-batchwise technology using egg-yolk phospholipids and the non-polar Amberlite resin. PIP1;1 proteoliposomes and control empty liposomes had good size homogeneity as seen by quasi-elastic light scattering and electron microscopy analyses. By stopped-flow light scattering, indicating correct protein folding of the incorporated protein, the osmotic water permeability exhibited by the PIP1;1 proteoliposomes was markedly higher than empty liposomes. Functional reconstitution of OsPIP1;1 was further confirmed by the low Arrhenius activation energy (3.37 kcal/mol) and sensitivity to HgCl2, a known AQP blocker, of the PIP1;1-mediated osmotic water conductance. These results provide a valuable contribution in fully elucidating the regulation and water-conducting property of PIP1;1, an AQP that needs to hetero-multimerize with AQPs of the PIP2 subgroup to reach the native plasma membrane and play its role. The micro-batchwise methodology is suitable for the functional reconstitution of whichever AQPs and other membrane transport proteins.

Keywords

Oryza sativa; AQP channels; PIP1; Osmotic water transport; Liposomes; Membrane protein reconstitution; Micro-batchwise technology

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...Acrylamide gels were stained with Coomassie Brilliant Blue (Sigma, St Louis, MO) or submitted to immunoblotting using an anti-6His tag monoclonal antibody (GenScript Corporation, Piscataway, NJ) at a final concentration of 0.2 μg/mL blocking buffer according to a previous...

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