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.
Takakura
Yoshimitsu
Y
Plant Innovation Center, Japan Tobacco, Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan. yoshimitsu.takakura@jt.com
Oka
Naomi
N
Kajiwara
Hitomi
H
Tsunashima
Masako
M
Usami
Satoru
S
Tsukamoto
Hiroshi
H
Ishida
Yuji
Y
Yamamoto
Takeshi
T
D001360:Avidin / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D055767:Immobilized Proteins / Q000378:metabolism*; D007700:Kinetics; D008863:Microspheres; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D030262:Soybean Proteins / Q000378:metabolism; D010835:Phytohemagglutinins
D001360:Avidin; D001710:Biotin; D002352:Carrier Proteins; D005656:Fungal Proteins; D055767:Immobilized Proteins; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D012799:Sialyltransferases; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins; C550582:tamavidin 2, Pleurotus cornucopiae