Conclusions

showed 2-fold higher absorbance change than the commercial HRP–pG, indicating signal amplification was achieved in ELISA using the HRP–pAG(biotin)–SA conjugate (Fig. 2-9D).

This result demonstrates the further modification of recombinant HRPs by MTG-mediated PTM. The modification of K-tags by MTG reaction can be achieved with using any molecule that contains a glutamine, including fluorescent molecules, azido or alkyne groups for click chemistry, as well as proteins. A peptide tag containing glutamine (Q-tag) could be used instead of the K-tag and a Q-tagged HRP may be a better candidate for modification with synthetic molecules because many amine-bearing functional molecules are already commercially available, but glutamine-bearing molecules are very limited. By introducing peptide tags that can be recognized by PTM enzymes [7] such as MTG and sortase A, recombinant HRPs with many functionalities can be engineered in a highly controlled manner, and the baculovirus–silkworm expression system is certainly one of the most feasible expression systems for producing recombinant HRPs.

conjugate showed 2-fold higher signal than the commercial HRP–pG conjugate, demonstrating the improvement of function of HRP–pAG by PTMs mediated by MTG. The recombinant production of HRP enabled us to fully utilize the function of HRP by combining genetic modification-based protein conjugation methods. By introducing peptide tags to achieve site-specific PTMs using enzymatic reactions or by fusing with protein functional domains, recombinant HRPs will become promising tools for biological applications.

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CHAPTER 3 FUNCTIONAL HORSERADISH PEROXIDASE−STREPTAVIDIN