Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties.
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.
Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. yoshimitsu.takakura@jt.com
Yoshimitsu
Y
Takakura
Naomi
N
Oka
Hitomi
H
Kajiwara
Masako
M
Tsunashima
D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D008297:Male; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D005656:Fungal Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C550582:tamavidin 2, Pleurotus cornucopiae; D001360:Avidin; D001710:Biotin; D019809:Streptavidin