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Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties

Paper ID Volume ID Publish Year Pages File Format Full-Text
20570 43181 2012 5 PDF Available
Title
Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties
Abstract

The avidin–biotin interaction is widely employed as a universal tool in numerous biotechnological applications. In avidin–biotin technology, non-specific binding to biological macromolecules is a hindrance. The major origin of this non-specific binding is the electrical charge of the surface of biotin-binding proteins. Tamavidin 2, a fungal avidin-like protein that binds biotin with an extremely high affinity, can be produced as a soluble recombinant protein in Escherichia coli. The isoelectric point of tamavidin 2 is 7.4–7.5, lower than avidin (10.0), and slightly higher than that of streptavidin (6.0–7.5). Here, we genetically engineered charge mutants of tamavidin 2 to reduce non-specific binding. By substituting an acidic residue (glutamic acid) for basic residues (arginine and lysine), we constructed three mutant proteins (muteins) and confirmed their high-level production in soluble form in E. coli, as well as that of tamavidin 2. We then tested these proteins for non-specific binding to salmon sperm DNA, glycoproteins (integrin and fibronectin), and IgG from human sera. The muteins showed lower non-specific binding than tamavidin 2 to these macromolecules. In particular, one mutein, tamavidin-R104EK141E, which had the lowest isoelectric point (5.8–6.2) among avidin, streptavidin and tamavidin 2, displayed the lowest non-specific binding. The affinity of this mutein to biotin was high, comparable with that of tamavidin 2. These findings indicate that tamavidin-R104EK141E has the potential to serve as a robust tool in the numerous applications of biotin-binding proteins.

Keywords
Avidin; Biotin-binding; Isoelectric point; Non-specific binding; Protein engineering; Tamavidin
First Page Preview
Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 114, Issue 5, November 2012, Pages 485–489
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering