Tamavidin 2-HOT, a highly thermostable biotin-binding protein

Yoshimitsu Takakura, Junko Suzuki, Naomi Oka, Yoshimitsu Kakuta

研究成果: ジャーナルへの寄稿記事

5 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)1-8
ページ数8
ジャーナルJournal of Biotechnology
169
発行部数1
DOI
出版物ステータス出版済み - 1 10 2014

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Biotin
Streptavidin
Avidin
Reducing agents
Escherichia coli
Fungal Proteins
Asparagine
Thermodynamic stability
Reducing Agents
Isoelectric Point
Dimethyl Sulfoxide
tamavidin 2-HOT
biotin-binding proteins
Carrier Proteins
Disulfides
Proteins
Cysteine
Molecules
Hot Temperature
Mutation

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

これを引用

Tamavidin 2-HOT, a highly thermostable biotin-binding protein. / Takakura, Yoshimitsu; Suzuki, Junko; Oka, Naomi; Kakuta, Yoshimitsu.

:: Journal of Biotechnology, 巻 169, 番号 1, 10.01.2014, p. 1-8.

研究成果: ジャーナルへの寄稿記事

Takakura, Yoshimitsu ; Suzuki, Junko ; Oka, Naomi ; Kakuta, Yoshimitsu. / Tamavidin 2-HOT, a highly thermostable biotin-binding protein. :: Journal of Biotechnology. 2014 ; 巻 169, 番号 1. pp. 1-8.
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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.",
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