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 (TPO₁₆₃) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO₁₆₃ 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 TPO₁₆₃-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 × 0.2 × 0.02 mm) grew in two space groups: P2₁, with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 Å, β = 90.24°, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 Å, β = 90.42°. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.