Naomi Oka

Tamavidin, a versatile affinity tag for protein purification and immobilization

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity and is highly produced in soluble form in Escherichia coli. By contrast, widely used biotin-binding proteins avidin and streptavidin are rarely produced in soluble form in E. coli. In this study, we describe an efficient system for one-step purification and immobilization of recombinant proteins using tamavidin 2 as an affinity tag. A bacterial sialyltransferase and soybean agglutinin were fused to tamavidin 2 and expressed in E. coli and tobacco BY-2 cells, respectively. High-level expressions of the fusion proteins were detected (80 mg l(-1)E. coli culture for bacterial sialyltransferase-tamavidin 2 and 2 mg l(-1) BY-2 cell culture for soybean agglutinin-tamavidin 2). To immobilize and purify the fusion proteins, biotinylated magnetic microbeads were incubated with the soluble extract from each recombinant host producing the fusion protein and then washed thoroughly. As the result, both fusion proteins were immobilized tightly on the microbeads without substantial loss of activity and simultaneously highly purified (90-95% purity) on the microbeads. Biotin with a longer linker contributed to higher affinity between the fusion protein and biotin. These results suggest that tamavidin fusion technology is a powerful tool for production, purification, and immobilization of recombinant proteins.

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom, Confers Resistance to the Blast Fungus Magnaporthe oryzae in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.

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

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.

Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity.