Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties |
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Authors: | Yoshimitsu Takakura Naomi Oka Hitomi Kajiwara Masako Tsunashima |
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Affiliation: | Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. Electronic address: yoshimitsu.takakura@jt.com. |
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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. |
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Keywords: | Avidin Biotin-binding Isoelectric point Non-specific binding Protein engineering Tamavidin |
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