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High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system

Paper ID Volume ID Publish Year Pages File Format Full-Text
34254 45013 2016 8 PDF Available
Title
High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system
Abstract

•Aquaporin Z (AqpZ) was expressed as the fusion protein maltose-binding protein (MBP)–AqpZ–polyhistidine (HIS).•The MBP tag of MBP–AqpZ–HIS was removed by factor Xa.•AqpZ–HIS was purified using affinity chromatography.•The yield of functional AqpZ–HIS was 15–20 mg/l.

Aquaporin Z (AqpZ) is a water channel protein belonging to the major intrinsic protein (MIP) family and is found in the membrane of Escherichia coli. Due to its high water permeability and low activation energy, AqpZ is a potential candidate for developing biomimetic water filtration technologies. In the present study, membrane-associated AqpZ was expressed in E. coli using the maltose-binding protein (MBP)/polyhistidine (HIS) dual-affinity tag fusion system. The effects of different host strains and the expression conditions on the production of the fusion protein MBP–AqpZ–HIS were systematically studied. Furthermore, an efficient protocol was developed to purify AqpZ–HIS via affinity chromatography, and approximately 15–20 mg of functional AqpZ–HIS was obtained from 1 l of culture with a specific water transport activity of around 2 × 10−14 cm3 s−1 monomer−1. The related sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and tryptophan fluorescence emission analyses indicated that purified AqpZ–HIS assembled as a homotetramer with high thermostability, in turn indicating the correct folding of AqpZ. Our results showed that the MBP/HIS dual-affinity tag system was highly efficient for the overexpression, membrane targeting, and purification of MIP channel proteins in E. coli.

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Keywords
Aquaporin Z; MBP/polyhistidine dual-affinity tag system; Membrane-associated expression; Membrane protein
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High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 51, Issue 5, May 2016, Pages 599–606
Authors
, , , , , , , , ,
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