Open AccessCrystallization of Spätzle, a cystine‐knot protein involved in embryonic development and innate immunity in Drosophila melanogaster
Author(s) -
Hoffmann Anita,
Neumann Piotr,
Schierhorn Angelika,
Stubbs Milton T.
Publication year - 2008
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309108018812
Subject(s) - crystallization , dimer , orthorhombic crystal system , crystallography , cystine , drosophila melanogaster , escherichia coli , chemistry , biology , biochemistry , gene , crystal structure , enzyme , organic chemistry , cysteine
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 Å resolution were obtained after three months by the sitting‐drop vapour‐diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 53.0, b = 59.2, c = 62.5 Å) that diffracted to 2.8 Å 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.