Large-Scale Purification of Functional Recombinant Human Aquaporin-2
| Title: | Large-Scale Purification of Functional Recombinant Human Aquaporin-2 |
|---|---|
| Authors: | Werten, Paul J.L.; Hasler, Lorenz; Koenderink, Jan B.; Klaassen, Corne H. W.; de Grip, Willem; Engel, Andreas; Deen, Peter M.T. |
| Publisher: | FEBS Letters |
| Date Published: | August 31, 2001 |
| Reference Number: | 517 |
The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
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In order to better understand the structure and function of AQP2, and thus to better provide therapeutic treatment or cures when AQP2 malfunctions, researchers must have available to them large amounts of AQP2 for structural analysis. However, as AQP2 only appears in the principal cells of the kidney collecting duct and the male gonad, isolating large amounts of AQP2 from natural sources is impossible. Werten, et al., successfully developed a way to obtain sufficient amounts of structurally and functionally accurate AQP2 for scientific study.
The researchers accomplished this by introducing genetically engineered AQP2 called HT-AQP2 (as it was tagged with a histidine amino acid marker) into a culture comprised of baculovirus and insect cells. Their method is able to produce 0.5 mg of pure HT-AQP2 per liter of their bioreactor culture. Their work should enable researchers to greatly further their understanding of AQP2.