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(1): 106-15, 2012 Mar.
Article
en En
| MEDLINE
| ID: mdl-22178733
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_neglected_diseases
/
3_zoonosis
Asunto principal:
Glutamato Carboxipeptidasa II
/
Antígenos de Superficie
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2012
Tipo del documento:
Article
País de afiliación:
República Checa