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In Vivo Site-Specific Incorporation of N-Acetyl-Galactosamine Amino Acids in Eubacteria

a technology of nacetylgalactosamine and amino acids, applied in the field of protein biochemistry, can solve the problems of difficult analysis of glycan structure and study of glycosylation effects on protein structure and function, and the method can become problemati

Inactive Publication Date: 2008-03-13
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural glycoproteins are often present as a population of many different glycoforms, which makes analysis of glycan structure and the study of glycosylation effects on protein structure and function difficult.
A limitation of this approach is that the primary glycosylation sites are predetermined by the cell line in which the protein is expressed.
32:6793-6796; and, Macmillan, D.; et al., (2002) Org Lett 4:1467-1470), but this method can become problematic with proteins that have more than one cysteine residue.

Method used

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  • In Vivo Site-Specific Incorporation of N-Acetyl-Galactosamine Amino Acids in Eubacteria
  • In Vivo Site-Specific Incorporation of N-Acetyl-Galactosamine Amino Acids in Eubacteria
  • In Vivo Site-Specific Incorporation of N-Acetyl-Galactosamine Amino Acids in Eubacteria

Examples

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example 1

System for Incorporating a Glycosylated Amino Acid into Proteins

[0294] In eukaryotes, mucins comprise the most prevalent type of O-glycans, constituting a polydisperse group of glycoproteins and proteoglycans involved in inflammation and cellular recognition (Hanisch (2001) Biol. Chem. 382:143-149). The N-acetylgalacosamine (GalNAc) saccharide α-linked to the hydroxyl group of threonine or serine represents the core unit (the “Tn antigen”) in mucin-type glycoproteins. A wide variety of mucin-type core structures are generated by subsequent glycosylation of the GalNAc residue at the C-3 and / or C-6 hydroxyl groups.

[0295] This example describes methods and compositions for preparing N-acetylgalactosamine-α-threonine (GalNAc-Thr) and co-translationally incorporating this unnatural amino acid into a protein in vivo. In order to genetically encode this functional group in E. coli in the form of GalNAc-threonine, a number of tRNA-synthetase pairs were evolved that are capable of insertin...

example 2

Strategy for the Synthesis of Glycoproteins

[0314] In another embodiment of the invention, homogeneous glycoproteins are synthesized in an organism, e.g., E. coli, by the cotranslational incorporation of the glycosylated amino acid N-acetylglucosamine-β-serine (GlcNAc-Ser). For example, myoglobin containing β-GlcNAc-serine at a defined position can be expressed in E. coli in good yield and with high fidelity. The β-GlcNAc moiety can be recognized by a carbohydrate binding protein or subsequently modified with a galactosyltransferase. This approach can also be applicable to other posttranslational modifications, e.g., protein phosphorylation, acetylation, methylation and the like.

[0315] Methods were previously developed which for the first time allowed the systematic addition of amino acids with novel chemical and physical properties to the genetic code of E. coli (see, e.g., L. Wang, et al., (2001) Science 292:498; L. Wang, et al., (2002) J. Am. Chem. Soc. 124:1836; Z. Zhang, et al...

example 3

Sequences of Exemplary O-RSs

[0328] Exemplary O-RSs that can be used in the invention include SEQ ID NOS.: 1-4 and 11-13 (See Table 3), and exemplary O-tRNA that can be used in the invention includes SEQ ID NO: 17. Exemplary polynucleotides that encode O-RSs include SEQ ID NOS.: 6-9 and 14-16.

[0329] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0330] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in va...

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Abstract

Methods and compositions for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid having a N-acetylgalactosamine moiety into a protein; optionally, the N-acetylgalactosamine-containing unnatural amino acid can be further modified with additional sugars.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and benefit of U.S. Provisional Patent Application Ser. No. 60 / 620,898, filed Oct. 20, 2004, the specification of which is incorporated herein in its entirety for all purposes.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under Grant No. GM44154 awarded by the National Institutes of Health. The Government may have certain rights to the invention.FIELD OF THE INVENTION [0003] The invention is in the field of protein biochemistry. The invention relates to compositions and methods for making and using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and pairs thereof, that incorporate unnatural amino acids into proteins, where the unnatural amino acids comprise an N-acetylgalactosamine moiety and the resulting proteins are glycoproteins. The invention also relates to methods of producing proteins...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/22C12N1/00C12N1/21C12P21/02C12N15/52C12N15/70
CPCC12N9/93C12P21/005C12N15/67
Inventor SCHULTZ, PETERHANSON, SARAHXU, RANZHANG, ZHIWENWONG, CHI-HUEY
Owner THE SCRIPPS RES INST
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