GENETICALLY ENCODED INITIATOR FOR POLYMER GROWTH FROM PROTEINS

摘要

This invention pertains to methods for producing homogeneous recombinant proteins that contain polymer initiators at defined sites. The unnatural amino acid, 4-(2'-bromoisobutyramido)phenylalanine of formula 1, was designed and synthesized as a molecule comprising a functional group further comprising an initiator for an atom-transfer radical polymerization (ATRP") that additionally would provide a stable linkage between the protein and growing polymer. We evolved a Methanococcus jannaschii (Mj) tyrosyl-tRNA synthetase/tRNAcuA pair to genetically encode this unnatural amino acid in response to an amber codon. To demonstrate the utility of this functional amino acid, we produced Green Fluorescent Protein with the unnatural amino acid initiator of formula 1 site- specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with oligo(ethylene oxide) mononiethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate wherein the polymer is connected at a specifically selected site on GFP.