Incorporation of glycosylated amino acid into protein by an in vitro translation system

Teruhiko Matsubara; Kazutoshi Iijima; Takayoshi Watanabe; Takahiro Hohsaka; Toshinori Sato

doi:10.1016/j.bmcl.2013.08.035

Incorporation of glycosylated amino acid into protein by an in vitro translation system

Teruhiko Matsubara, Kazutoshi Iijima, Takayoshi Watanabe, Takahiro Hohsaka, Toshinori Sato

Department of Biosciences and Informatics

Research output: Contribution to journal › Article › peer-review

8 Citations (Scopus)

Abstract

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

Original language	English
Pages (from-to)	5634-5636
Number of pages	3
Journal	Bioorganic and Medicinal Chemistry Letters
Volume	23
Issue number	20
DOIs	https://doi.org/10.1016/j.bmcl.2013.08.035
Publication status	Published - 2013 Oct 15

Keywords

Expanding genetic code
Glycoprotein
Glycosylated amino acid
In vitro translation system
O-Glycan

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Molecular Biology
Pharmaceutical Science
Drug Discovery
Clinical Biochemistry
Organic Chemistry

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title = "Incorporation of glycosylated amino acid into protein by an in vitro translation system",

abstract = "O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.",

keywords = "Expanding genetic code, Glycoprotein, Glycosylated amino acid, In vitro translation system, O-Glycan",

author = "Teruhiko Matsubara and Kazutoshi Iijima and Takayoshi Watanabe and Takahiro Hohsaka and Toshinori Sato",

note = "Funding Information: This work was partly supported by a grant for Keio Leading-edge Laboratory of Science and Technology (KLL) specified research projects (T.M.) and by the Global Center-of-Excellence Program for Human Metabolomic Systems Biology (Keio University) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT)(K.I.). ",

year = "2013",

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doi = "10.1016/j.bmcl.2013.08.035",

language = "English",

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AU - Matsubara, Teruhiko

AU - Iijima, Kazutoshi

AU - Watanabe, Takayoshi

AU - Hohsaka, Takahiro

AU - Sato, Toshinori

N1 - Funding Information: This work was partly supported by a grant for Keio Leading-edge Laboratory of Science and Technology (KLL) specified research projects (T.M.) and by the Global Center-of-Excellence Program for Human Metabolomic Systems Biology (Keio University) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT)(K.I.).

PY - 2013/10/15

Y1 - 2013/10/15

N2 - O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

AB - O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

KW - Expanding genetic code

KW - Glycoprotein

KW - Glycosylated amino acid

KW - In vitro translation system

KW - O-Glycan

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Incorporation of glycosylated amino acid into protein by an in vitro translation system

Abstract

Keywords

ASJC Scopus subject areas

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