Abstract
Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.
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Acknowledgments
pICH41414 and pICH51288 were a gift from Sylvestre Marillonnet & Nicola Patron (Addgene plasmid # 50337 and Addgene plasmid # 50269, respectively). EC15259 was a gift from the ENSA consortium. This project was financially supported by BBSRC 18RM1 project BB/S003193/1.
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Schuster, M., Paulus, J.K., Kourelis, J., van der Hoorn, R.A.L. (2022). Purification of His-Tagged Proteases from the Apoplast of Agroinfiltrated N. benthamiana . In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_5
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DOI: https://doi.org/10.1007/978-1-0716-2079-3_5
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