title: A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins.
abstract: A universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of not more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.
authors.affiliation: Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Friedrich
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Hoheisel
authors.forename: Jens-Peter
authors.initials: JP
authors.lastname: Knemeyer
authors.forename: Nicole
authors.initials: N
authors.lastname: Marmé
mesh_terms: D001483:Base Sequence; D043422:Carboxypeptidases A / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D004247:DNA; D001616:beta-Galactosidase; D043422:Carboxypeptidases A