University of California

The Scripps Research Institute

Franklin and Marshall College

Ryan Aaron Mehl

Lei Wang

Peter G Schultz

David R Liu

John Christopher Anderson

Thomas J Magliery

Eric L Meggers

Miro Pastrnak

Zhiwen Zhang

Jason W Chin

Stephen William Santoro

Steven William Santoro

Jason William Chin

Krzysztof A Matyjaszewski

Saadyah Averick

Miro Pastranak

Peter Schultz

The present invention is directed towards a protein-polymer composition having a protein with a site-specifically incorporated unnatural amino acid initiator and a covalently attached polymer.

Site specifically incorporated initiator for growth of polymers from proteins

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(Mj) tyrosyl-tRNA synthetase/tRNApair 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)monomethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate wherein the polymer is connected at a specifically selected site on GFP.

Genetically encoded initiator for polymer growth from proteins

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

The invention provides methods and compositions for in vivo incorporation of unnatural amino acids. Also provided are compositions including proteins with unnatural amino acids.

In vivo incorporation of unnatural amino acids

Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

This invention relates, in part, to newly identified polynucleotides, polypeptides, variants and derivatives thereof; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; and uses of the polynucleotides, polypeptides, variants and derivatives. The invention also relates to compositions of orthogonal aminoacyl-tRNA synthetases, and pairs of orthogonal aminoacyl-tRNA synthetases, and orthogonal tRNAs that incorporate fluorinated amino acids into proteins in response to selector codons. The present invention also includes translation biochemistry methods for site-specific incorporation of fluorinated amino acids, for example,F- orF-labelled amino acids, into proteins or peptides. Such amino acids may be used as an NMR probe for characterizing protein structure, dynamics, and reactivity or for radionuclide imaging (e.g., PET). Fluorinated amino acids may also be used to stabilize proteins or peptides.

Site-specific incorporation of fluorinated amino acids into proteins

Growing community of inventors

Lancaster, PA, United States of America

Ryan Aaron Mehl