Production of O-GlcNAc modified recombinant proteins in Escherichia coli

Ki Hong Lim, Chang Hoon Ha, Hyo-Ihl Chang

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O-GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

Original languageEnglish
Pages (from-to)306-311
Number of pages6
JournalJournal of Microbiology and Biotechnology
Volume12
Issue number2
Publication statusPublished - 2002 Jan 1

Fingerprint

Recombinant proteins
Recombinant Proteins
Escherichia coli
Maltose-Binding Proteins
Proteins
Maltose
Eukaryotic Cells
Fusion reactions
Sp1 Transcription Factor
Glycosylation
Transcription factors
Acetylglucosamine
Purification
Catalyst activity
Plasmids
O-GlcNAc transferase
Carrier Proteins

Keywords

  • Cotransformation
  • O-GlcNAc transferase
  • O-linked N-acetylglucosamine

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Production of O-GlcNAc modified recombinant proteins in Escherichia coli. / Lim, Ki Hong; Ha, Chang Hoon; Chang, Hyo-Ihl.

In: Journal of Microbiology and Biotechnology, Vol. 12, No. 2, 01.01.2002, p. 306-311.

Research output: Contribution to journalArticle

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AB - O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O-GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

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