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Crystallization of Spatzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster.


ABSTRACT: The Spätzle 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 A) that diffracted to 2.8 A resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle 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.

SUBMITTER: Hoffmann A 

PROVIDER: S-EPMC2494967 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster.

Hoffmann Anita A   Neumann Piotr P   Schierhorn Angelika A   Stubbs Milton T MT  

Acta crystallographica. Section F, Structural biology and crystallization communications 20080705 Pt 8


The Spätzle 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 cond  ...[more]

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