| 摘要 |
<p>An in vitro method for producing a semi-synthetic fusion protein is provided, whereby a target protein fused to an intein - a protein splicing element - is selectively cleaved in a first step as depicted in Figure 1 with a thiol reagent, forming a carboxyl-terminal thioester of the target protein and releasing the target protein from the intein. In a subsequent step as shown in Figure 1, a desired, synthetic, protein or peptide having an amino-terminal cysteine is ligated to the target protein. Standard thiol-reagents such as DTT, or thiol-reagents optimized for ligation such as the odorless MESNA, may be used in the first step. The method permits the direct ligation of a desired peptide to a thioester bond that had linked a target protein to an intein. An in vivo variation of the method will permit production of a cytotoxic protein: a truncated, inactive, form of the protein fused to an intein is introduced in vivo, this fusion product is then selectively cleaved, and a synthetic protein or peptide is subsequently ligated at a carboxyl-terminal thioester of the target protein in order to restore the native activity of the cytotoxic protein.</p> |
