| 摘要 |
Patent No. 596259 Disclosed 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 by a lantibiotic enzyme capable of forming a thioether bridge between residues X1 and X2, wherein X1 is selected from the group consisting of Dhb, Dha, Thr, and Ser and wherein X2 is Cys or Lys; or wherein X1 is Cys or Lys and X2 is selected from the group consisting of Dhb, Dha, Thr and Ser; Tag is an amino acid sequences serving as affinity tag when cyclized, said affinity tag allowing for capture of the proteinaceous substance to a specific binding partner of the tag; and wherein said motif is preceded N-terminally by a lantibiotic leader sequence; b. Contacting said precursor with lantibiotic enzyme LanM, cyclase LanC in the case of a combination of a dehydroresidue and a cysteine, or a combination of a lantibiotic dehydratase LanB and cyclase LanC in case of X1 or X2, thereby introducing an intramolecular ring structure comprising the Tag sequence; and c. Isolating the resulting cyclized proteinaceous substances. Also disclosed is a proteinaceous substance comprising at least one cyclic tag sequence, wherein said cyclic tag sequence is a cyclized streptavidin binding sequence, the tag sequence being part of a thioether-linked ring structure bridging (in the orientation N- to C-) a D-amino acid and an L-amino acid, or an L-amino acid to a D amino acid.
|
