US 8,012,739 B2Granted

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

AssigneesThe Scripps Research Institute, The Regents of the University of California

InventorsPeter G. Schultz, Lei Wang, John Christopher Anderson, Jason W. Chin, David R. Liu, Thomas J. Magliery, Eric L. Meggers, Ryan Aaron Mehl, Miro Pastrnak, Stephen William Santoro, Zhiwen Zhang

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Abstract

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.

Claims12

Dependent claims

A cell comprising an orthogonal tRNA synthetase (ORS) and an orthogonal tRNA (OtRNA), as a pair wherein:

(a) said ORS preferentially aminoacylates said OtRNA, wherein preferential aminoacylation is defined in that said ORS aminoacylates endogenous tRNAs of the cell with a reduced efficiency as compared to endogenous tRNA synthetase aminoacylation of said endogenous tRNAs of the cell;
(b) said ORS preferentially aminoacylates said OtRNA with an unnatural amino acid, as compared to any natural amino acid;
(c) said OtRNA is aminoacylated by the endogenous tRNA synthetases of the cell with reduced efficiency as compared to aminoacylation of the endogenous tRNAs by said endogenous tRNA synthetases;
(d) said OtRNA recognizes a selector codon of an mRNA in said cell;
(e) kcat/Km for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than kcat/Km for aminoacylation of the OtRNA by the ORS with the natural amino acid, or wherein said unnatural amino acid is incorporated into a growing polypeptide in the cell with a fidelity of greater than 75% in response to the selector codon;
and,
(f) said cells are cultured in the presence of said unnatural amino acid.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 11/978,154Filed 26 Oct 2007 · continuation
Priority: 19 Apr 2001
Published: US 2009/0227002 A110 Sep 2009
Granted: 6 Sep 2011
Term ends: Not estimatedThe source records don’t support an estimate.

Classification17 codes

C12P 21/00
Preparation of peptides or proteins
A61P 7/00
Drugs for disorders of the blood or the extracellular fluid
A61P 43/00
Drugs for specific purposes, not provided for in groups a61p1/00-a61p41/00
C07K 14/00
Peptides having more than 20 amino acids

Family

Application60/285,030Us provisional application · filed 19 Apr 2001
Application60/355,514Us provisional application · filed 6 Feb 2002
Application10/126,931Parent of this continuation · filed 19 Apr 2002
Application11/254,170Parent of this continuation · filed 18 Oct 2005
US 2009/0227002 A1Pre-grant publication · published 10 Sep 2009
US 7,354,761 B2Parent of this continuation
US 7,083,970 B2Parent of this continuation
US 2003/0108885 A1Published application
US 2006/0234367 A1Published application
US 8,012,739 B2This patent

From the US filing history. Foreign family members aren’t listed.