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

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2299-2304
Number of pages6
JournalBiomacromolecules
Volume6
Issue number4
DOIs
Publication statusPublished - Jul 1 2005

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Biomaterials
  • Polymers and Plastics
  • Materials Chemistry

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