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

• We developed a biotin-binding protein with high thermostability (tamavidin 2-HOT).
• Tamavidin 2-HOT contained engineered inter-subunit disulfide bridges.
• Tamavidin 2-HOT was active after heating at 99.9 °C for 30 min and in 70% DMSO.
• Tamavidin 2-HOT bound biotin strongly and was produced efficiently in Escherichia coli.
• Tamavidin 2-HOT may be useful for biotin-binding under harsh assay conditions.

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 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 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.