3RBU

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

Summary for 3RBU

• Entry DOI: 10.2210/pdb3rbu/pdb
• Descriptor: Glutamate carboxypeptidase 2, 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, ... (10 entities in total)
• Functional Keywords: peptidase, biotinylation, extracellular, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens (human)
• Cellular location: Cell membrane; Single-pass type II membrane protein. Isoform PSMA': Cytoplasm: Q04609
• Total number of polymer chains: 1
• Total formula weight: 86480.67

Authors

Tykvart, J.,Sacha, P.,Barinka, C.,Starkova, J.,Knedlik, T.,Lubkowski, J.,Konvalinka, J. (deposition date: 2011-03-30, release date: 2012-01-04, Last modification date: 2023-09-13)

Primary citation

Tykvart, J.,Sacha, P.,Barinka, C.,Knedlik, T.,Starkova, J.,Lubkowski, J.,Konvalinka, J.
Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.
Protein Expr.Purif., 82:106-115, 2012

PubMed Abstract: 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.

PubMed: 22178733
DOI: 10.1016/j.pep.2011.11.016

Experimental method

X-RAY DIFFRACTION (1.6 Å)