Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production

Biotechnol J. 2018 Jul;13(7):e1800050. doi: 10.1002/biot.201800050. Epub 2018 Mar 26.


Medicago truncatula is an established model for studying legume biology. More recently, it has also been exploited as a Molecular Farming platform for the production of recombinant proteins, with the successful expression of fungal and human proteins in plants and cell suspension cultures of this species. One of the challenges that now must be overcome is the degradation of final products during production and downstream processing stages. In the M. truncatula genome, there are more than 400 putative protease-encoding genes, but to date, the proteolytic content of Medicago cell cultures has not been studied. In this report, the proteolytic activities that can potentially hamper the successful production of recombinant proteins in this system are evaluated. The potential proteases responsible for the degradation of target proteins are identified. Interestingly, the number of proteases found in Medicago spent medium is considerably lower than that of the well-established tobacco bright yellow 2 (BY-2) system. Papain-like cysteine proteases are found to be the major contributors to recombinant protein degradation in Medicago. This knowledge is used to engineer a cell line with reduced endogenous protease activity by expressing a selective protease inhibitor, further improving this expression platform.

Keywords: ABPP; BY-2; Medicago truncatula; cystatin; proteases.

MeSH terms

  • Cell Culture Techniques
  • Cell Engineering
  • Medicago truncatula* / enzymology
  • Medicago truncatula* / genetics
  • Medicago truncatula* / metabolism
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / metabolism*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Recombinant Proteins / analysis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transfection


  • Plant Proteins
  • Recombinant Proteins
  • Peptide Hydrolases