Purification of His-Tagged Proteases from the Apoplast of Agroinfiltrated N. benthamiana

Methods Mol Biol. 2022;2447:53-66. doi: 10.1007/978-1-0716-2079-3_5.


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.

Keywords: Agroinfiltration; His-tag; Immobilized metal affinity chromatography; Nicotiana benthamiana; Protease; Protein purification; Recombinant protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Affinity / methods
  • Endopeptidases
  • Peptide Hydrolases* / metabolism
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Recombinant Proteins
  • Tobacco* / genetics
  • Tobacco* / metabolism


  • Plant Proteins
  • Recombinant Proteins
  • Endopeptidases
  • Peptide Hydrolases