Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor

We present the concept of chemo-enzymic backbone engineering of proteins. Recombinant DNA techniques are used to produce appropriate proteins that are enzymically fragmented to give the starting materials. These fragments are modified specifically at their chain termini either enzymically (coupling of a hydrazide to the C terminus) or chemically (periodate oxidation of N-terminal serine to a glyoxylyl function). The modified fragments, which need no side protection whatever, are mixed together and religate themselves spontaneously under mild conditions. The hydrazone bond thus formed can be reduced if desired, which stabilizes the linkage and enhances the flexibility of the local conformation. In this way biologically or chemically derived structures can be incorporated into the protein, and the choice of the chemical ones is free of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be derived by either recombinant or chemical methods alone. We illustrate the particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony-stimulating factor. Analogs constructed include one which, in spite of having a nonpeptide link in its backbone, has full biological activity.