A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins
| Autor: | Jens-Peter Knemeyer, Jörg D. Hoheisel, Achim Friedrich, Nicole Marmé |
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| Rok vydání: | 2011 |
| Předmět: |
Base Sequence
Carboxypeptidases A Staining and Labeling Oligonucleotide Chemistry Proteins DNA beta-Galactosidase Sensitivity and Specificity Biochemistry Enzyme Activation Protein structure Affinity chromatography FLAG-tag Covalent bond Labelling Escherichia coli Molecule Target protein Hydrophobic and Hydrophilic Interactions Molecular Biology Fluorescent Dyes |
| Zdroj: | PROTEOMICS. 11:3757-3760 |
| ISSN: | 1615-9853 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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