Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster.
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.
Hoffmann
Anita
A
Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
Neumann
Piotr
P
Schierhorn
Angelika
A
Stubbs
Milton T
MT
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003460:Crystallization; D029721:Drosophila Proteins / Q000302:isolation & purification / Q000502:physiology*; D004331:Drosophila melanogaster / Q000196:embryology* / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D047108:Embryonic Development / Q000502:physiology*; D007113:Immunity, Innate / Q000502:physiology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029721:Drosophila Proteins; C086179:spz protein, Drosophila