Journal
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS
Volume 64, Issue -, Pages 707-710Publisher
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S1744309108018812
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The Spatzle protein is involved in both the definition of the dorsal-ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 A resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals ( space group P2(1)2(1)2(1), with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 angstrom) that diffracted to 2.8 angstrom resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spatzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.
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