Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Jo Tominaga, Noriho Kamiya, Satoshi Doi, Hirofumi Ichinose, Tatsuo Maruyama, Masahiro Goto

研究成果: ジャーナルへの寄稿記事

41 引用 (Scopus)

抄録

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with β-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position of the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (kcat) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

元の言語英語
ページ(範囲)2299-2304
ページ数6
ジャーナルBiomacromolecules
6
発行部数4
DOI
出版物ステータス出版済み - 7 1 2005

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Enzyme immobilization
Transglutaminases
Phosphatases
Immobilization
Peptides
Alkaline Phosphatase
Sepharose
Casein
Enzymes
Caseins
glycyl-seryl-alanine
Immobilized Proteins
Proteins
Glutamine
Escherichia coli
Lysine
Gels

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Biomaterials
  • Polymers and Plastics
  • Materials Chemistry

これを引用

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase. / Tominaga, Jo; Kamiya, Noriho; Doi, Satoshi; Ichinose, Hirofumi; Maruyama, Tatsuo; Goto, Masahiro.

:: Biomacromolecules, 巻 6, 番号 4, 01.07.2005, p. 2299-2304.

研究成果: ジャーナルへの寄稿記事

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abstract = "Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with β-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position of the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (kcat) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.",
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AU - Tominaga, Jo

AU - Kamiya, Noriho

AU - Doi, Satoshi

AU - Ichinose, Hirofumi

AU - Maruyama, Tatsuo

AU - Goto, Masahiro

PY - 2005/7/1

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N2 - Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with β-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position of the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (kcat) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

AB - Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with β-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position of the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (kcat) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

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