CHEMOENZYMATIC 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 protei ns. Recombinant DNA techniques are used to produce appropriate protein s that are enzymically fragmented to give the starting materials. Thes e fragments are modified specifically at their chain termini either en zymically (coupling of a hydrazide to the C terminus) or chemically (p eriodate 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 stabil izes the linkage and enhances the flexibility of the local conformatio n. In this way biologically or chemically derived structures can be in corporated into the protein, and the choice of the chemical ones is fr ee of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be der ived by either recombinant or chemical methods alone. We illustrate th e particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony stimulating f actor. Analogs constructed include one which, in spite of having nonpe ptide link in its backbone, has full biological activity.