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Tamavidin 2-REV: An engineered tamavidin with reversible biotin-binding capability ☆

Paper ID Volume ID Publish Year Pages File Format Full-Text
23556 43452 2013 7 PDF Available
Title
Tamavidin 2-REV: An engineered tamavidin with reversible biotin-binding capability ☆
Abstract

A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8–4.4 × 10−7 M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules.

► Tamavidin 2-REV is a novel biotin-binding protein with reversible biotin-binding. ► The productivity of tamavidin 2-REV in Escherichia coli was very high. ► A biotinylated protein was efficiently purified by using tamavidin 2-REV. ► Tamavidin 2-REV was resistant to protease K and it had neutral isoelectric point. ► Tamavidin 2-REV is a powerful tool for purification of biotinylated biomolecules.

Keywords
Affinity purification; Avidin; Biotinylated protein; Reversible biotin-binding; Tamavidin
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Tamavidin 2-REV: An engineered tamavidin with reversible biotin-binding capability ☆
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 1, 10 March 2013, Pages 19–25
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