title: Tamavidin 2-HOT, a highly thermostable biotin-binding protein.
abstract: Tamavidin 2 is a fungal tetrameric protein that binds with high affinity to biotin, like avidin and streptavidin. We replaced asparagine-115, which lies in a subunit-subunit interface of tamavidin 2, with cysteine to generate the novel, highly thermostable protein tamavidin 2-HOT. Tamavidin 2-HOT retained more than 80% of its biotin-binding activity even after incubation at 99.9°C for 60min and was fully active in 70% dimethylsulfoxide for 30min, whereas in these harsh conditions, avidin, streptavidin, and tamavidin 2 lost their activities (less than 20% of their biotin-binding activities). The Tm in which the biotin-binding activity becomes half of tamavidin 2-HOT was 105°C, at least 20°C higher than those of avidin, streptavidin, and tamavidin 2. Because a reducing agent removed the thermal stability of tamavidin 2-HOT, the N115C mutation likely created disulfide bridges that stabilized inter-subunit associations. Tamavidin 2-HOT is efficiently produced in the soluble form by Escherichia coli for practical use. The isoelectric point of tamavidin 2-HOT (7.4) is sufficiently low to reduce the chance for non-specific binding of non-target molecules due to high positive charges. Therefore, tamavidin 2-HOT may be useful in diverse novel applications that take advantage of its high biotin-binding capability that can withstand harsh conditions.
authors.affiliation: Plant Innovation Center, Japan Tobacco, Inc. , 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. Electronic address: yoshimitsu.takakura@jt.com.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Takakura
authors.affiliation: Plant Innovation Center, Japan Tobacco, Inc. , 700 Higashibara, Iwata, Shizuoka 438-0802, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Plant Innovation Center, Japan Tobacco, Inc. , 700 Higashibara, Iwata, Shizuoka 438-0802, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Oka
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Kakuta
mesh_terms: D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; C017071:biotin-binding proteins; C000590533:tamavidin 2-HOT; D004121:Dimethyl Sulfoxide
keywords: Biotin-binding; Dimethylsulfoxide; Disulfide bridge; Subunit association; Thermal stability