title: Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody.
abstract: Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.
authors.affiliation: Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. r-kuroki@kirin.co.jp
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
authors.forename: Masako
authors.initials: M
authors.lastname: Hirose
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kato
authors.forename: Michael D
authors.initials: MD
authors.lastname: Feese
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.forename: Hideki
authors.initials: H
authors.lastname: Shigematsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Watarai
authors.forename: Yoshitake
authors.initials: Y
authors.lastname: Maeda
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Tahara
authors.forename: Takashi
authors.initials: T
authors.lastname: Kato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyazaki
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D003460:Crystallization; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology*; D046911:Macromolecular Substances; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013926:Thrombopoietin / Q000737:chemistry* / Q000276:immunology*; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D013926:Thrombopoietin