Journal of Biotechnology 169 (2014) 1–8 Contents lists available at ScienceDirect Journal of Biotechnology jo u r n al homep age: www.elsevier.com/locate/jbiotec Tamavidin 2-HOT, a highly thermostable biotin-binding protein  Yoshimitsu Takakura a,∗,1 , Junko Suzuki a , Naomi Oka a , Yoshimitsu Kakuta b a Plant Innovation Center, Japan Tobacco, Inc. , 700 Higashibara, Iwata, Shizuoka 438-0802, Japan b Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan a r t i c l e i n f o Article history: Received 16 July 2013 Received in revised form 17 October 2013 Accepted 25 October 2013 Available online 7 November 2013 Keywords: Biotin-binding Dimethylsulfoxide Disulfide bridge Subunit association Thermal stability a b s t r a c t Tamavidin 2 is a fungal tetrameric protein that binds with high affinity to biotin, like avidin and strepta- vidin. 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 60 min and was fully active in 70% dimethylsulfoxide for 30 min, whereas in these harsh conditions, avidin, streptavidin, and tamavidin 2 lost their activities (less than 20% of their biotin-binding activities). The T m in which the biotin-binding activity becomes half of tamavidin 2-HOT was 105 ◦ C, at least 20 ◦ C higher than those of avidin, strep- tavidin, 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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Avidin, which originates from chicken, and its bacterial ana- log, streptavidin, which comes from Streptomyces avidinii, are homotetrameric proteins that bind biotin with very high affin- ity (K d of 6 × 10 -16 M and 4 × 10 -14 M, respectively), the highest known ligand–protein affinities known in nature (Green, 1990). This characteristic of the interaction between biotin and avidin or streptavidin has great technical value and is a useful tool in diverse life-science fields in numerous biochemical, pharmaceutical, bio- physical, and biotechnological applications (Laitinen et al., 2007; Schetters, 1999; Wilchek and Bayer, 1990). Many applications of the high-affinity binding of biotin involve the study of nucleic acids. Typically, biotinylated single-stranded nucleotides are immobilized onto (strept)avidin- bearing solid phases through the (strept)avidin–biotin interaction (Sabanayagam et al., 2000; Sassolas et al., 2008). Because single- stranded oligo DNAs and RNAs occasionally form stable hairpin  Note: Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession number AB827410 for tam2-N115C. ∗ Corresponding author. Tel.: +81 285 34 2655; fax: +81 285 25 4460. E-mail address: yoshimitsu.takakura@jt.com (Y. Takakura). 1 Present address: Leaf Tobacco Research Center, Japan Tobacco, Inc., 1900 Idei, Oyama, Tochigi 323-0808, Japan. structures with T m s ranging from 80 ◦ C to more than 90 ◦ C (Hirao et al., 1992; Kaushik et al., 2003; Tuerk et al., 1988), the strong sec- ondary structure of polynucleotides might block interactions with other molecules. Therefore, if experiments can be done at temper- atures high enough to denature nucleic acid (for example, 95 ◦ C or greater), the nucleotides might be immobilized efficiently without steric hindrance. Other substances of interest may be hydrophobic and therefore soluble only in organic solvents. Because avidin and streptavidin are inactive near and at boiling temperatures and in organic solvents, novel biotin-binding proteins that remain stable under such extreme conditions are highly desirable. The numerous factors involved in thermal stability of proteins offer many approaches to engineering thermo-stable forms. One key factor in the stability of proteins is inter-subunit association (Akanuma et al., 1999; Goodsell and Olson, 2000; Ivens et al., 2002; Matsumura et al., 1989). Thus, the amino acid residues involved in subunit–subunit interaction can be potential targets in modify- ing biotin-binding proteins for higher stability. This approach was tested in the replacement of isoleucine-117 and histidine-127 in the subunit–subunit interfaces of avidin and streptavidin, respec- tively, with cysteine (Nordlund et al., 2003; Reznik et al., 1996); the resulting proteins (avidin-I117C and streptavidin-H127C) were significantly more tolerant to higher temperatures than were the original proteins. In these cases, inter-subunit disulfide bridges were formed, and the newly formed bridges prevented dissocia- tion of tetramers at a high temperature. However, these muteins 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.10.034