Methods of incorporating amino acid analogs into proteins

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

STATEMENT OF GOVERNMENT SUPPORT[0001]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.REFERENCE TO RELATED APPLICATION[0002]This application is a continuation of U.S. patent application Ser. No. 11 / 130,583, filed May 17, 2005, now pending, which 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.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 macromolecu...

AI Technical Summary

Benefits of technology

[0009]Compositions of the present invention include an orthogonal aminoacyl-tRNA synthetase (O-RS), where the O-RS preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid, optionally, in vivo. In one embodiment, the invention provides a nucleic acid encoding an O-RS, or a complementary nucleic acid sequence thereof. In another embodiment, the O-RS has improved or enhanced enzymatic properties, e.g., the Km is higher or lower, the kcat is higher or lower, the value of kcat / Km is higher or lower or the like, for the unnatural amino acid compared to a naturally occurring amino acid, e.g., one of the 20 known amino acids.

[0012]In certain embodiments, the modified tRNA can be efficiently charged to carry an analog of the natural amino acid (e.g. the unnatural amino acid).

[0022]In certain embodiments, the modified tRNA further comprises a mutation at the fourth, extended anticodon site for increase translational efficiency.

[0089]In certain embodiments, the polypeptide, when PEGylated, has one or more of: longer half life, sustained or enhanced biological activity, is homogeneously modified, increased potency and stability and / or decreased immunogenicity, consistency in biological activities from lot to lot.

[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 any of the suitable subject methods, wherein the unnatural amino acid(s) reduces binding affinity of the cytokine or growth factor to its receptor in endosomes, thereby increasing the half-life of the cytokine or growth factor.

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 suppression efficiency will interfere with the normal translation termination of some non-targeted proteins in the organism.

On the other hand, a low suppression efficiency will likely be insufficient to suppress more than one nonsense or frame-shift mutation sites in the target protein, such that it becomes more and more difficult or impractical to synthesize a full-length target protein incorporating more and more non-canonical amino acids.

Although this method provides efficient incorporation of analogues at multiple sites, it suffers from the limitation that the novel amino acid must “share” codons with one of the natural amino acids.

This may be undesirable, since for an engineered enzyme or protein, non-canonical amino acid incorporation at an unintended site may unexpectedly compromise the function of the protein, while missing incorporating the non-canonical amino acid at the designed site will fail to achieve the design goal.

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