High-resolution structure of a fish aquaporin reveals a novel extracellular fold

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High-resolution structure of a fish aquaporin reveals a novel extracellular fold
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Quick-closing valve allows fish to rapidly regulate the water in their cells

Yeast clone selection was performed on YPD plates containing 2,000 μg/ml Zeocin , and small-scale protein production was tested for selected strains. Membrane fractions were analyzed by SDS–PAGE and immunoblot. Strains with the highest production of His-tagged proteins were picked for large-scale production in 3-liter fermentors. For untagged-cpAQP1aa-243, the strain corresponding to the largest colony on the high-Zeocin plate was used for fermentation directly.

Membrane solubilization of cpAQP1aa-FL was carried out by mixing the membrane with solubilization buffer in a 1:1 volume ratio supplemented with Complete EDTA-free protease inhibitor . The mixture was gently rocked for 1 h in a cold room. Unsolubilized material was removed by ultracentrifugation , and the supernatant was transferred to a falcon tube containing pre-equilibrated Ni-NTA agarose beads and rocked for 3 h or overnight.

For the untagged protein, the solubilization buffer contained 20 mM Tris–HCl , 10% glycerol, 4% NG, and 2 mM β-MeOH. After solubilization and centrifugation, the solubilized material was diluted in a 1:3 ratio with dropwise addition of pre-chilled dilution buffer . The diluted sample was filtered using a 0.2-μm bottle top filter and applied to a Resource S cation exchange column pre-equilibrated with buffer A at a flow rate of 0.5 ml/min.

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