Purification of His-tagged proteases from the apoplast of agroinfiltrated N. benthamiana

Mariana Schuster, Judith K. Paulus, Jiorgos Kourelis, Renier A. L. van der Hoorn

Research output: Chapter in Book/Report/Conference proceedingChapter


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.

Original languageEnglish
Title of host publicationPlant Proteases and Plant Cell Death
Subtitle of host publicationMethods and Protocols
EditorsMarina Klemenčič, Simon Stael, Pitter F. Huesgen
PublisherHumana Press Inc
Number of pages14
ISBN (Electronic)978-1-0716-2079-3
ISBN (Print)978-1-0716-2078-6, 978-1-0716-2081-6
Publication statusPublished - 19 May 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Agroinfiltration
  • His-tag
  • Immobilized metal affinity chromatography
  • Nicotiana benthamiana
  • Protease
  • Protein purification
  • Recombinant protein

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