Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes

Patmawati xxxx, Kosuke Minamihata, Tsuneyuki Tatsuke, Jae Man Lee, Takahiro Kusakabe, Noriho Kamiya

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

Original languageEnglish
Article number1700624
JournalBiotechnology Journal
Volume13
Issue number6
DOIs
Publication statusPublished - Jun 2018

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

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