title: Expression and functional characterization of recombinant tributyltin-binding protein type 2.
abstract: Tributyltin-binding proteins (TBT-bps) are members of the fish lipocalins that were isolated from the blood of Japanese flounder (Paralichthys olivaceus) and function in the binding and detoxification of TBT. In this study, we constructed a baculovirus-silkworm expression system and obtained recombinant TBT-bp2 (rTBT-bp2; 31.5 kDa) from the hemolymph of silkworm larvae injected with a recombinant baculovirus containing the TBT-bp2 gene. The binding potential of rTBT-bp2 was investigated and compared to that of the previously available recombinant TBT-bp1 (rTBT-bp1). Both rTBT-bp2 and rTBT-bp1 bound to DAUDA, a typical fluorescent ligand of lipocalins, with dissociation constants of 0.97 and 1.75 µM, respectively. The Hill coefficient value indicated that rTBT-bp2 may have multiple binding sites and strong negative cooperativity. These results suggest that the typical central cavity of lipocalins composed of eight specific β-sheets is conserved in rTBT-bp2, as it is in rTBT-bp1, although rTBT-bp2 has different effects than rTBT-bp1 in TBT binding. In a competition assay, rTBT-bp2 displayed exponential binding affinity to TBT with an inhibition constant of 0.29 µM, demonstrating that TBT binds to the central ligand pocket of rTBT-bp2. However, three fatty acids did not show any affinity to rTBT-bp2. Further studies are required to elucidate the endogenous function of TBT-bps as fish lipocalins and their function in responding to xenobiotics.
authors.affiliation: Laboratory of Marine Biochemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo.
authors.forename: Hina
authors.initials: H
authors.lastname: Satone
authors.forename: Eriko
authors.initials: E
authors.lastname: Akahoshi
authors.forename: Aiko
authors.initials: A
authors.lastname: Nakamura
authors.forename: Jae Man
authors.initials: JM
authors.lastname: Lee
authors.forename: Masato
authors.initials: M
authors.lastname: Honda
authors.forename: Yohei
authors.initials: Y
authors.lastname: Shimasaki
authors.forename: Shun-Ichiro
authors.initials: S
authors.lastname: Kawabata
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kusakabe
authors.forename: Yuji
authors.initials: Y
authors.lastname: Oshima
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D012831:Bombyx / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005432:Flounder; D018014:Gene Transfer Techniques; D006458:Hemolymph; D007814:Larva; D054834:Lipocalins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins; D014220:Trialkyltin Compounds / Q000378:metabolism* / Q000633:toxicity
chemical_list: D029941:Fish Proteins; D054834:Lipocalins; D011994:Recombinant Proteins; D014220:Trialkyltin Compounds; C011559:tributyltin