Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody.
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.
Kuroki
Ryota
R
Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. r-kuroki@kirin.co.jp
Hirose
Masako
M
Kato
Yoichi
Y
Feese
Michael D
MD
Tamada
Taro
T
Shigematsu
Hideki
H
Watarai
Hiroshi
H
Maeda
Yoshitake
Y
Tahara
Tomoyuki
T
Kato
Takashi
T
Miyazaki
Hiroshi
H
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
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D013926:Thrombopoietin