Eukaryotic expression, purification, crystallization and preliminary X-ray analysis of murine Manic Fringe.
Fringe proteins are Golgi-resident beta1,3-N-acetylglucosaminyltransferases that regulate development in metazoa through glycosylation of the Notch receptor and its ligands. The catalytic domain of murine Manic Fringe was expressed in the baculovirus/insect-cell system as a secreted protein. Mass-spectrometric analysis of the purified protein indicated the presence of two N-linked glycans. Abolishing the glycosylation sites by site-directed mutagenesis was necessary in order to obtain orthorhombic crystals that diffracted to 1.8 angstroms resolution. For phasing, a highly redundant data set was collected using a crystal soaked with halide salts.
Jinek
Martin
M
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Conti
Elena
E
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D005964:Glucosyltransferases; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015398:Signal Transduction; D014961:X-Ray Diffraction
D011506:Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C486343:Mfng protein, mouse