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

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. Genetic modification of proteins can provide novel functional proteins with desired functions. In this study, the baculovirus‐silkworm expression system is used for production of a fusion protein of horseradish peroxidase (HRP) and antibody binding proteins (HRP‐pAG). HRP‐pAG is expressed in a soluble apo form in silkworm and is activated by incubating with hemin. HRP‐pAG possesses a peptide tag containing lysine (K‐tag) at the C‐terminus and it can be modified with biotin using microbial transglutaminase reaction. The biotinylated HRP‐pAG forms conjugates with streptavidin and it shows higher signals than the commercially available HRP‐protein G conjugate in enzyme‐linked immunosorbent assay. 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.