Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• 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 Escherlchia coil. The isoelectnc 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. coil, 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-R104EKI41E has the potential to serve as a robust tool in the numerous applications of biotin-binding proteins.

著者

• Takakura Yoshimitsu

  Plant Breeding And Genetics Research Laboratory Japan Tobacco Inc.:(present Address)plant Innovation

• Kajiwara Hitomi

  Plant Innovation Center, Japan Tobacco, Inc.

• Tsunashima Masako

  Plant Innovation Center, Japan Tobacco, Inc.

• Oka Naomi

  Plant Innovation Center, Japan Tobacco, Inc.

関連論文

• Purification, Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete, Tricholoma matsutake(Biochemistry & Molecular Biology)
• Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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