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Preparative scale production and functional reconstitution of a human aquaglyceroporin (AQP3) using a cell free expression system

Paper ID Volume ID Publish Year Pages File Format Full-Text
33341 44971 2013 7 PDF Available
Title
Preparative scale production and functional reconstitution of a human aquaglyceroporin (AQP3) using a cell free expression system
Abstract

•We establish an efficient cell-free strategy to produce AQP3, an aquaglyceroporin.•Milligrams quantities of high quality AQP3 are made in a single reaction, in ml volumes.•The developed cell-free system is reliable, easily scalable and flexible.•Other aquaglyceroporins could be made at preparative scale by the devised strategy.•The proposed system may accelerate high-throughput studies and translational studies.

Understanding the selectivity of aquaporin (AQP) membrane channels and exploiting their biotechnological potential will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of high quality membrane proteins. Here, we establish a convenient method to synthesize large amounts of functional human aquaglyceroporin 3 protein (AQP3), an AQP of physiological relevance conducting glycerol and some small neutral solutes besides water. Milligram amounts of AQP3 were produced as a histidine-tagged protein (hAQP3-6His) in an Escherichia coli extract-based CF system in the presence of the non-ionic detergent Brij-98. The recombinant AQP3 was purified by affinity chromatography, incorporated into liposomes and evaluated functionally by stopped-flow light scattering. Correct protein folding was indicated by the high glycerol and water permeability exhibited by the hAQP3-6His proteoliposomes as compared to empty control liposomes. Functionality of hAQP3-6His was further confirmed by the strong inhibition of the glycerol and water permeability by phloretin and HgCl2, respectively, two blockers of AQP3. Fast and convenient CF production of functional AQP3 may serve as basis for further structural/functional assessment of aquaglyceroporins and help boosting the AQP-based biomimetic technologies.

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Preparative scale production and functional reconstitution of a human aquaglyceroporin (AQP3) using a cell free expression system
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Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 30, Issue 5, 25 June 2013, Pages 545–551
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us