3RBU: N-Terminally Avitev-Tagged Human Glutamate Carboxypeptidase Ii In Complex With 2-Pmpa

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.
PDB ID: 3RBUDownload
MMDB ID: 96222
PDB Deposition Date: 2011/3/30
Updated in MMDB: 2012/12
Experimental Method:
x-ray diffraction
Resolution: 1.6  Å
Source Organism:
Similar Structures:
Biological Unit for 3RBU: dimeric; determined by author and by software (PISA)
Molecular Components in 3RBU
Label Count Molecule
Proteins (2 molecules)
Glutamate Carboxypeptidase 2(Gene symbol: FOLH1)
Molecule annotation
Chemicals (28 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB