3rbu | pdb_00003rbu

N-terminally AviTEV-tagged Human Glutamate Carboxypeptidase II in complex with 2-PMPA

spinqualitylabelsall models 3rbu, resolution 1.60Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3rbu is a 1 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: BMA, CA, CL, G88, NAG, ZN, MAN Gene: FOLH, FOLH1, GIG27, NAALAD1, PSM, PSMA (Homo sapiens) Activity: Glutamate carboxypeptidase II, with EC number 3.4.17.21 Resources: FirstGlance, OCA, RCSB, PDBsum Publication Abstract from PubMed Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.,Tykvart J, Sacha P, Barinka C, Knedlik T, Starkova J, Lubkowski J, Konvalinka J Protein Expr Purif. 2011 Dec 8. PMID:22178733 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Carboxypeptidase

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