Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides


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Erbakan M. , Shen Y., Grzelakowski M., Butler P. J. , Kumar M., Curtis W. R.

PLOS ONE, vol.9, no.1, 2014 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 9 Issue: 1
  • Publication Date: 2014
  • Doi Number: 10.1371/journal.pone.0086830
  • Title of Journal : PLOS ONE

Abstract

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (similar to 20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17 +/- 0.05 cm/s) and low energy of activation for water transport (2.93 +/- 0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (+/- 0.41) x 10(-12) cm(3)/s or 4.17 (+/- 1.38) x 10(10) H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.