Molecular cloning, overexpression and characterization of a novel water channel protein from Rhodobacter sphaeroides.
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 (∼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)×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.
Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Mustafa
M
Erbakan
Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Yue-xiao
YX
Shen
AquaZ Inc, Cincinnati, Ohio, United States of America.
Mariusz
M
Grzelakowski
Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Peter J
PJ
Butler
Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Manish
M
Kumar
Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America ; Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Wayne R
WR
Curtis
D000465:Algorithms; D000595:Amino Acid Sequence; D020346:Aquaporins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D008081:Liposomes / Q000378:metabolism / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014867:Water / Q000378:metabolism
D020346:Aquaporins; D001426:Bacterial Proteins; D008081:Liposomes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014867:Water