title: Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.
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.
authors.affiliation: Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Tykvart
authors.forename: P
authors.initials: P
authors.lastname: Sácha
authors.forename: C
authors.initials: C
authors.lastname: Bařinka
authors.forename: T
authors.initials: T
authors.lastname: Knedlík
authors.forename: J
authors.initials: J
authors.lastname: Starková
authors.forename: J
authors.initials: J
authors.lastname: Lubkowski
authors.forename: J
authors.initials: J
authors.lastname: Konvalinka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D018360:Crystallography, X-Ray; D004330:Drosophila / Q000166:cytology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D011994:Recombinant Proteins; D001710:Biotin; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II