Methods of incorporating amino acid analogs into proteins

a technology proteins, applied in the field of amino acid analogs in proteins, can solve the problems of limiting the chemical functionality of the method, unable to circumvent unable to achieve the specificity of the synthetase, so as to increase reduce the binding affinity, and enhance the half-life of the cytokine or growth factor

Inactive Publication Date: 2005-12-29
CALIFORNIA INST OF TECH
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Benefits of technology

[0091] Another aspect of the invention provides a method for enhancing half-life of a cytokine or a growth factor, comprising incorporating one or more unnatural amino acid(s) at specified position(s) of the polypeptide using

Problems solved by technology

Though this process has been very useful for designing new macromolecules with precise control of composition and architecture, a major limitation is that the mutagenesis is restricted to the 20 naturally occurring amino acids.
Nevertheless, the number of amino acids shown conclusively to exhibit translational activity in vivo is small, and the chemical functionality that has been accessed by this method remains modest.
In designing macromolecules with desired properties, this poses a limitation since such designs may require incorporation of complex analogs that differ significantly from the natural substrates in terms of both size and chemical properties and hence, are unable to circumvent the specificity of the synthetases.
However, their utility in multisite incorporation is limited by modest (20-60%) suppression efficiencies (Anderson et al., J. Am. Chem. Soc.
This is so partially because too high a stop codon suppressio

Method used

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  • Methods of incorporating amino acid analogs into proteins
  • Methods of incorporating amino acid analogs into proteins
  • Methods of incorporating amino acid analogs into proteins

Examples

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examples

[0496] This invention is further illustrated by the following examples which should not be construed as limiting. The teachings of all references, patents and published patent applications cited throughout this application, as well as the Figures are hereby incorporated by reference.

example i

tRNA and Synthetase Construction

[0497] This example illustrates the incorporation of an amino acid analog in proteins at positions encoded by codons which normally encode phenylalanine (Phe). A schematic diagram is shown in FIG. 1. Similar approaches can be used for any other analogs.

[0498] Phe is encoded by two codons, UUC and UUU. Both codons are read by a single tRNA, which is equipped with the anticodon sequence GAA. The UUC codon is therefore recognized through standard Watson-Crick base-pairing between codon and anticodon; UUU is read through a G-U wobble base-pair at the first position of the anticodon (Crick, J. Mol. Biol. 19: 548, 1966; Soll and RajBhandary, J. Mol. Biol. 29: 113, 1967). Thermal denaturation of RNA duplexes has yielded estimates of the Gibbs free energies of melting of G-U, G-C, A-U, and A-C basepairs as 4.1, 6.5, 6.3, and 2.6 kcal / mol, respectively, at 37° C. Thus the wobble basepair, G-U, is less stable than the Watson-Crick basepair, A-U. A modified tR...

example ii

Generation of a Mutant Protein Containing NaI

[0503] Murine dihydrofolate reductase (mDHFR), which contains nine Phe residues, was chosen as the test protein. The expression plasmid pQE16 encodes MDHFR under control of a bacteriophage T5 promoter; the protein is outfitted with a C-terminal hexahistidine (HIS6) tag to facilitate purification via immobilized metal affinity chromatography.

[0504] In this construct, four of the Phe residues of mDHFR are encoded by UUC codons, five by UUU. A full-length copy of the mu-yPheRS gene, under control of a constitutive tac promoter, was inserted into pQE16. The gene encoding ytRNAPheAAA was inserted into the repressor plasmid pREP4 (Qiagen) under control of the constitutive promoter lpp. E. coli transformants harboring these two plasmids were incubated in Phe-depleted minimal medium supplemented with 3 mM NaI and were then treated with 1 mM IPTG to induce expression of mDHFR. Although the E. coli strain (K10-F6) used in this study is a Phe auxo...

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Abstract

The invention provides a method of incorporating nonstandard amino acids into a protein by utilizing a modified aminoacyl-tRNA synthetase to charge the nonstandard amino acid to a modified tRNA, which forms strict Watson-Crick base-pairing with a codon that normally forms wobble base-pairing with natural tRNAs.

Description

REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of the filing date of U.S. Provisional Application 60 / 571,810, filed on May 17, 2004, the entire content of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT [0002] This invention was made with federal government support under grant number GM62523 awarded by the NIH, and under NSF DMR-0080065 awarded by the NSF. The United States government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Protein engineering is a powerful tool for modification of the structural catalytic and binding properties of natural proteins and for the de novo design of artificial proteins. Protein engineering relies on an efficient recognition mechanism for incorporating mutant amino acids in the desired protein sequences. Though this process has been very useful for designing new macromolecules with precise control of composition and architecture, a major limitation is that the mutagenesi...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07K14/00C12N1/18C12N1/21C12N9/00C12N15/74C12P21/02C12P21/06
CPCC12N9/93C12P21/02C07H21/02C12N2310/10C12P21/00C12N15/113C12N15/70
Inventor KWON, INCHANTIRRELL, DAVID
Owner CALIFORNIA INST OF TECH
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