AMINO ACID SUBSTITUTED MOLECULES
First Claim
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1. A method for producing a modified target polypeptide, comprising:
- (a) providing a host cell, the host cell comprising a vector having a polynucleotide encoding the target polypeptide,(b) site-specifically incorporating one or more non-natural amino acid codons into the polynucleotide, wherein at least one non-natural amino acid codon corresponds to the first position of the amino terminus of the target polypeptide,(c) growing the host cell under conditions such that the host cell expresses the modified target polypeptide, wherein the modified target polypeptide comprises a non-natural amino acid residue at the first position of the amino terminus, and wherein the non-natural amino acid residue comprises an azide, alkyne, vinyl, or aryl halide group, thereby producing a modified target polypeptide.
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Abstract
The invention provides compositions and methods of identifying, modifying and producing modified target molecules, including therapeutic molecules by modification with non-natural amino acids. Certain aspects of the invention include methods of adding a chemical moiety to a target molecule, and the compositions resulting therefrom. Certain aspects of the invention also relate to kits for identifying, modifying and producing modified target molecules described herein.
96 Citations
49 Claims
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1. A method for producing a modified target polypeptide, comprising:
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(a) providing a host cell, the host cell comprising a vector having a polynucleotide encoding the target polypeptide, (b) site-specifically incorporating one or more non-natural amino acid codons into the polynucleotide, wherein at least one non-natural amino acid codon corresponds to the first position of the amino terminus of the target polypeptide, (c) growing the host cell under conditions such that the host cell expresses the modified target polypeptide, wherein the modified target polypeptide comprises a non-natural amino acid residue at the first position of the amino terminus, and wherein the non-natural amino acid residue comprises an azide, alkyne, vinyl, or aryl halide group, thereby producing a modified target polypeptide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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- 13. A composition comprising a modified target polynucleotide encoding a modified target polypeptide, the modified target polynucleotide comprising one or more non-natural amino acid codons that encode at least one non-natural amino acid comprising an azide, alkyne, vinyl, or aryl halide group and corresponding to the first position of the amino terminus of the target polypeptide.
- 17. A modified target polypeptide comprising one or more non-natural amino acid residues, wherein at least one of the non-natural amino acid residues is the first position of the amino terminus, wherein the non-natural amino acid residue comprises an azide, alkyne, vinyl, or aryl halide group.
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24. A method for preparing a refolded, soluble form of an insoluble or aggregated PEGylated interferon beta protein comprising one or more free cysteine residues, said method comprising the steps of:
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(a) causing a host cell to express a interferon beta protein in an insoluble or aggregated form; (b) lysing the cells by chemical, enzymatic or physical means; (c) solubilizing the insoluble or aggregated protein by exposing the insoluble or aggregated protein to a denaturing agent, a reducing agent and a cysteine blocking agent; (d) PEGylating the reduced, denatured protein; and (e) refolding the PEGylated protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the PEG interferon beta protein to refold into a soluble, biologically active form.
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25. A method for producing a modified target polypeptide, comprising:
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(a) providing a host cell comprising a vector, wherein said vector comprises a polynucleotide encoding a target polypeptide; (b) site-specifically incorporating one or more non-natural amino acid residue(s) into the target polypeptide to produce a modified target polypeptide, wherein; (i) one or more codons encoding naturally occurring amino acids are incorporated into said polynucleotide; (ii) the one or more non-natural amino acid(s) are incorporated into the target polypeptide at one or more natural amino acid position(s) encoded by the one or more codon(s) of (i) or one or more unchanged codon(s) encoding a natural amino acid; and (c) growing the host cell under conditions such that the host cell expresses the modified target polypeptide. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33)
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34. A method of modifying a polypeptide containing a non-natural amino acid, comprising incorporating a chemical moiety into said polypeptide by a chemical reaction selected from the group consisting of:
- palladium-catalyzed Suzuki coupling, palladium-catalyzed Sonogashira coupling, palladium-catalyzed Heck coupling, palladium-catalyzed Hiyama coupling and Huisgen copper-catalyzed [3+2] cycloaddition, in the presence of micelles or DTT, or both, wherein said modified polypeptide retains its native function.
- View Dependent Claims (35, 36, 37)
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38. A modified human interferon-13 polypeptide comprising the following amino acid alterations:
- methionine at position 1 to azidohomoalanine, serine at position 2 to glutamic acid, cysteine at position 17 to serine, methionine at position 36 to isoleucine, isoleucine at position 40 to phenylalanine, isoleucine at position 44 to leucine, methionine at position 62 to isoleucine, and methionine at position 117 to threonine.
- View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
Specification