Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097

Abstract

The secreted cystine-knot protein sclerostin was first identified from genetic screening of patients suffering from the rare bone-overgrowth diseases sclerosteosis and van Buchem disease. Sclerostin acts a negative regulator of bone growth through inhibiting the canonical Wnt signalling cascade by binding to and blocking the Wnt co-receptor LRP5/6. Its function in blocking osteoblastogenesis makes it an important target for osteoanabolic therapy approaches to treat osteoporosis, which is characterized by a progressive decrease in bone mass and density. In this work, the production, crystallization and preliminary X-ray diffraction data analysis of a sclerostin-neutralizing human Fab antibody fragment, AbD09097, obtained from a naive antibody library are reported. Crystals of the Fab AbD09097 belonged to space group P21, with unit-cell parameters a = 45.19, b = 78.49, c = 59.20 Å, β = 95.71° and diffracted X-rays to a resolution of 1.8 Å.

Keywords: Wnt signalling pathway; bone homeostasis; osteoporosis; sclerostin.

Figure 1

(a) Rod-shaped crystals of the Fab AbD09097. The crystals grew to maximal dimensions of 200 × 50 × 50 µm within 7 d. (b) SDS–PAGE and subsequent Western blot analysis of crystals as shown in (a) using an anti-His₆ antibody confirmed the presence of full-length Fab protein with the Myc and His₆ tag attached to the C-terminus of the Fab heavy chain.

Figure 2

Diffraction image of a crystal of AbD09097 (as shown in Fig. 1 ▶). The crystals diffracted to 1.9 Å resolution (exposure time 150 s) as seen from the enlargement in the upper left inset.