title: Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability.
abstract: A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8-4.4×10(-7)M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules.
authors: Plant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. yoshimitsu.takakura@jt.com
authors: Yoshimitsu
authors: Y
authors: Takakura
authors: Kozue
authors: K
authors: Sofuku
authors: Masako
authors: M
authors: Tsunashima
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001360:Avidin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D055767:Immobilized Proteins; D011994:Recombinant Proteins; C550582:tamavidin 2, Pleurotus cornucopiae; D001360:Avidin; D012710:Serum Albumin, Bovine; D012694:Serine; D001710:Biotin; D000409:Alanine