Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants
We previously described a procedure for the use of plant protease inhibitors as “companion” accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265–273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along—and co-migrating—with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.
KeywordsClinically useful recombinant proteins Heterologous protein expression Recombinant protein degradation Companion protease inhibitors Protein stabilization
- 24.Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 77:e50521Google Scholar
- 28.Goulet M-C, Dallaire C, Vaillancourt L-P, Khalf M, Badri MA, Preradov A, Duceppe M-O, Goulet C, Cloutier C, Michaud D (2008) Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites. Plant Physiol 146:1010–1019CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Smith BJ (1984) SDS polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) Methods in molecular biology, vol. 1 Proteins. Humana, Clifton, NJ, pp 41–55Google Scholar
- 34.Gooderham K (1984) Transfer techniques in protein blotting. In: Walker JM (ed) Methods in molecular biology, vol. 1 Proteins. Humana, Clifton, NJ, pp 165–178Google Scholar
- 40.Pinkhasov J, Alvarez ML, Rigano MM, Piensook K, Larios D, Pabst M, Grass J, Mukherjee P, Gendler SJ, Walmsley AM, Mason HS (2011) Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice. Plant Biotechnol J 9:991–1001CrossRefPubMedGoogle Scholar