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Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II
- 1.0377067 - ÚOCHB 2013 RIV US eng J - Journal Article
Tykvart, Jan - Šácha, Pavel - Bařinka, Cyril - Knedlík, Tomáš - Starková, Jana - Lubkowski, J. - Konvalinka, Jan
Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.
Protein Expression and Purification. Roč. 82, č. 1 (2012), s. 106-115. ISSN 1046-5928. E-ISSN 1096-0279
R&D Projects: GA MŠMT 1M0508; GA MŠMT LC512
Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701
Keywords : affinity purification * biotin acceptor peptide * recombinant protein expression * biotin-protein ligase (BirA) * co-localization * PSMA
Subject RIV: CE - Biochemistry
Impact factor: 1.429, year: 2012
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.
Permanent Link: http://hdl.handle.net/11104/0215606
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