A protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast Charles Goulet 1,† , Moustafa Khalf 1 , Frank Sainsbury 1 , Marc-Andre ´ D’Aoust 2 and Dominique Michaud 1,* 1 De ´partement de phytologie, Universite ´ Laval, Pavillon des Services (INAF), Que ´bec, QC, Canada 2 Medicago, Inc., Que ´bec, QC, Canada Received 17 February 2011; revised 22 June 2011; accepted 28 June 2011. *Correspondence (fax +1 418 656 7856; email dominique.michaud@fsaa.ulaval.ca) † Present address: Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. Keywords: cell secretory pathway, leaf apoplast proteome, molecular farming, Nicotiana benthamiana, pro- teases, protease inhibitors, recombi- nant proteins, transient expression. Summary Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then per- formed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys prote- ases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1 ⁄ S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by 45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presum- ably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. Introduction The growing demand for recombinant therapeutic and diagnos- tic proteins worldwide (Leader et al., 2008), and the structure of these proteins often requiring complex post-translational maturation (Walsh and Jefferis, 2006; Gomord et al., 2010) strongly contribute to the rising popularity of plant protein expression platforms (Faye and Gomord, 2010). Plants offer obvious advantages as protein biofactories in terms of cost effi- ciency, product safety, flexible scalability and competence to perform post-translational modifications typical of mammalian proteins (Karg and Kallio, 2009). Many recombinant proteins of medical interest have been successfully expressed in plant sys- tems over the last 20 years, including a variety of blood pro- teins, hormones, growth regulators, enzyme inhibitors, vaccine antigens and mammalian antibodies (Ma et al., 2003; Daniell et al., 2009; De Muynck et al., 2010; Rybicki, 2010). Major pro- gress has been achieved over the years towards the optimiza- tion of transgene transcription and translation in plant systems, the development of highly efficient expression platforms and the understanding of protein post-translational modifications specific to the plant cell machinery (Potenza et al., 2004; Faye et al., 2005; Gleba et al., 2007; Streatfield, 2007; Daniell et al., 2009; Sainsbury et al., 2009; Gomord et al., 2010). A key challenge, at this stage, is to protect the integrity and overall quality of the recombinant protein products. Unlike sev- eral less complex pharmaceuticals, proteins present a natural tendency to structure heterogeneity, often giving a complex mixture of protein variants differing in their primary or tertiary structure (Faye et al., 2005). One factor strongly influencing the accumulation of recombinant proteins is their inherent instabil- ity in heterologous environments (Doran, 2006; Benchabane et al., 2008). Proteolytic enzymes are involved in numerous vital processes in plants (Schaller, 2004; van der Hoorn, 2008), but their abundance in plant tissues often represents a burden to the effective heterologous production of proteins. Whereas a number of recombinant proteins are accumulated under a sta- ble form in plant systems, several others undergo partial or extensive hydrolysis negatively impacting their final yield and quality (Benchabane et al., 2008). In particular, studies reported the unintended processing of recombinant proteins along the cell secretory pathway by resi- dent proteases of the ER, the Golgi or the apoplast (e.g. Ste- vens et al., 2000; Sharp and Doran, 2001; Outchkourov et al., 2003; Schiermeyer et al., 2005; Badri et al., 2009a; Benchabane et al., 2009; De Muynck et al., 2009). A well-known example of this is the systematic processing of mammalian antibodies in plant systems (Gomord et al., 2004; De Muynck et al., 2010). ª 2011 The Authors Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 83 Plant Biotechnology Journal (2012) 10, pp. 83–94 doi: 10.1111/j.1467-7652.2011.00643.x