Biologically produced cyclic affinity tags

The invention relates to novels ways of introducing an affinity tag into a protein of interest. Provided is an enzymatic method for providing a proteinaceous substance comprising a polypeptide of interest and a cyclic affinity tag, comprising the steps of: (a) providing at least one precursor proteinaceous substance, the precursor comprising said protein of interest and at least one motif of the general formula X1-Tag-X2, wherein X1 and X2 represent amino acids whose side chains can be linked enzymatically by a covalent bond; Tag is a short amino acid sequence capable of binding to a binding partner of the tag when cyclized; (b) contacting said precursor with at least one enzyme capable of forming a covalent bond between X1 and X2, thereby introducing an intramolecular ring structure comprising the Tag sequence; and (c) isolating the resulting cyclized proteinaceous substance. Also provided are proteinaceous substances obtainable thereby and the use thereof, for instance for preparing a peptide library.