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

• US 20090227002A1
• Filed: 10/26/2007
• Published: 09/10/2009
• Est. Priority Date: 04/19/2001
• Status: Active Grant

• Current Assignee: the united states of america as represented by the secretary of the navy
• Original Assignee: Scripps Research Institute and The Regents of The University of California
• Inventors: Magliery, Thomas J., Mehl, Ryan Aaron, Schultz, Peter G., Wang, Lei, Pastrnak, Miro, Chin, Jason W., Santoro, Stephen William, Liu, David R., Zhang, Zhiwen, Anderson, John Christopher, Meggers, Eric L.
• Granted Patent: US 8,012,739 B2
• Time in Patent Office: Days
• US Class Current: 435/252.330
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 13/00 Preparation of nitrogen-containing organic compounds
• CPC Class Codes: C12P 13/005 Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12P 13/22 Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

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.

Claims

1-115. -115. (canceled)

116. 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; and

wherein k_cat/K_m for aminoacylation of the OtRNA by the ORS with the unnatural amino acid is higher than k_cat/K_m 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

(e) said cells are cultured in the presence of said unnatural amino acid.

117. The cell of claim 116, wherein said unnatural amino acid is selected from a group consisting of:

O-methyl-L-tyrosine, L-3-(2-naphthyl)-alanine, p-benzoyl-L-phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, p-amino-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, and isopropyl-L-phenylalanine.

118. The cell of claim 116, wherein the unnatural amino acid is p-azido-L-phenylalanine.

119. The cell of claim 116, wherein the unnatural amino acid is p-acetyl-L-phenylalanine.

120. The cell of claim 116, wherein the unnatural amino acid is m-acetyl-L-phenylalanine.

121. The cell of claim 116, wherein the unnatural amino acid is 4-(2-oxo-propoxy)-L-phenylalanine.

122. The cell of claim 116, wherein said cell is a prokaryotic cell.

123. The cell of claim 122 wherein said cell is a bacterium.

124. The cell of claim 122 wherein said cell is an Escherichia coli cell.

125. The cell of claim 116, wherein said selector codon is an amber codon or a four base codon.

126. The cell of claim 116, wherein the orthogonal tRNA synthetase comprises an amino acid sequence as set forth in SEQ ID No.:

62.

127. The cell of claim 116, wherein the orthogonal tRNA is encoded by a nucleic acid comprising a sequence as set forth in SEQ ID No.:

1.