Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases

Pedro Castanheira, Carla Almeida, Daniela Dias-Pedroso, Isaura Simões

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Number of pages13
Publication statusPublished - 2022

Publication series

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


  • Aspartic proteases
  • Atypical aspartic proteases
  • E. coli
  • Heterologous expression
  • Inclusion bodies
  • Nucellin-like aspartic proteases
  • Pepsin-like
  • Plant
  • Refolding
  • Typical aspartic proteases


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