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Author Takakura, Yoshimitsu ♦ Oka, Naomi ♦ Kajiwara, Hitomi ♦ Tsunashima, Masako
Source World Health Organization (WHO)-Global Index Medicus
Content type Text
Publisher Elsevier
File Format HTM / HTML
Language English
Difficulty Level Medium
Subject Domain (in DDC) Natural sciences & mathematics ♦ Chemistry & allied sciences ♦ Life sciences; biology ♦ Physiology & related subjects ♦ Biochemistry ♦ Natural history of organisms ♦ Microorganisms, fungi & algae ♦ Technology ♦ Medicine & health ♦ Human physiology ♦ Pharmacology and therapeutics ♦ Diseases ♦ Chemical engineering ♦ Manufacture for specific uses ♦ Precision instruments & other devices
Subject Domain (in MeSH) Eukaryota ♦ Bacteria ♦ Organisms ♦ Heterocyclic Compounds ♦ Amino Acids, Peptides, and Proteins ♦ Chemicals and Drugs ♦ Investigative Techniques ♦ Analytical, Diagnostic and Therapeutic Techniques and Equipment ♦ Chemical Phenomena ♦ Biological Sciences
Subject Keyword Discipline Biomedical Engineering ♦ Discipline Microbiology ♦ Avidin ♦ Chemistry ♦ Metabolism ♦ Carrier Proteins ♦ Fungal Proteins ♦ Genetics ♦ Biotin ♦ Escherichia Coli ♦ Humans ♦ Isoelectric Point ♦ Male ♦ Mutant Proteins ♦ Protein Binding ♦ Protein Engineering ♦ Recombinant Proteins ♦ Streptavidin ♦ Journal Article
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.
Description Country affiliation: Japan
Author Affiliation: Takakura Y ( Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. yoshimitsu.takakura@jt.com)
ISSN 13891723
Educational Role Student ♦ Teacher
Age Range above 22 year
Educational Use Reading ♦ Research ♦ Self Learning
Interactivity Type Expositive
Education Level UG and PG
Learning Resource Type Article
Publisher Date 2012-11-01
Publisher Place Japan
e-ISSN 13474421
Journal Journal of Bioscience and Bioengineering
Volume Number 114
Issue Number 5


Source: WHO-Global Index Medicus