Protein sequestration in, or targeting to, specific cell compartments has also been readily recognized as a key factor determining the overall stability and yield of recombinant proteins in planta (Wandelt et al., 1992; Schouten et al., 1996; Gomord et al., 1997). Organelles play specific, complementary functions in the cell and thus harbour their own metabolic machinery, including a protease complement well adapted to their particular enzymatic and physicochemical environment (Callis, 1995). Not surprisingly, the targeting of foreign proteins to different organelles using appropriate peptide signals has a strong impact on their accumulation rate (Table 1). In biochemical terms, the stability of a given protein in vivo will depend on the relative steric accessibility of peptides or peptide strings on the protein chain susceptible to the proteases present. In practice, the choice of a suitable cellular destination will also depend on the structural characteristics of the recombinant protein, which will often dictate specific co- or post-translational modifications essential for adequate activity, stability and/or homogeneity (Faye et al., 2005).
Eventual metabolic interference effects on the host plant actively expressing the recombinant protein should also be assessed, given the possible onset of organelle-dependent effects of biologically active proteins in planta. Cytosolic or apoplastic accumulation of the human growth factor, for instance, had toxic effects in leaves of Nicotiana benthamiana, whereas, in contrast, no negative effects were observed for the same protein targeted to the chloroplast (Gils et al., 2005). Likewise, the bovine protease inhibitor aprotinin was accumulated at high specific levels when retained in the endoplasmic reticulum (ER) of potato leaf cells, but this positive effect of ER retention was counterbalanced by a general decrease in total protein content in leaves, presumably as a result of the exogenous inhibitor affecting key steps of protein biosynthesis in vivo (Badri, 2006). Considering the complexity of protein maturation processes in plant cells and the often unpredictable nature of pleiotropic effects in transgenic host plants, the most appropriate way to select a suitable cellular destination at this stage involves the empirical testing of different possible destinations, taking into account current knowledge on the protein being expressed and on the physicochemical and enzymatic microenvironment of the different organelles available for protein accumulation. Several subcellular compartments have been considered as possible destinations for recombinant proteins in plant cells, including the cytosol, the chloroplast and different subcompartments of the cell secretory pathway (Ma et al., 2003; Daniell, 2006; Goulet and Michaud, 2006).
Retention in the cytosol
In practice, the absence of a targeting signal in the transgene sequence prevents migration of the recombinant protein out of the cytosol following mRNA translation (Figure 1). Recombinant proteins retained in the cytosol are usually detected at very low levels despite good transgene transcription rates, giving, in several cases, accumulation rates below 0.1% of TSP (Conrad and Fiedler, 1998). Cytosolic targeting of the tomato mosaic virus antibody ‘rAb29’ in tobacco leaf cells, for instance, resulted in very weak accumulation rates, whereas the same transgene including a signal peptide for extracellular secretion produced easily detectable amounts of this protein (Schillberg et al., 1999). Similarly, retaining human growth hormone in the cytosol of N. benthamiana leaf cells led to protein levels of about 0.01% of TSP, in contrast with concentrations reaching 10% of TSP for the same protein targeted to the apoplast (Gils et al., 2005). Several factors may explain the limited suitability of the cytosol as a destination for recombinant proteins: (i) the negative redox potential of the cytosolic milieu, unfavourable to the correct folding of proteins with disulphide bonds (Goulet and Michaud, 2006); (ii) the absence of important co- and post-translational modification processes, such as glycosylation, which may have a positive impact on the folding, assembly and/or structural stability of several nascent and mature proteins (Faye et al., 2005); and (iii) the effective housekeeping activity of the ubiquitin–proteasome proteolytic pathway in this cellular compartment (Vierstra, 1996, 2003), involved notably in the recognition and degradation of incorrectly folded proteins. Although some recombinant proteins remain stable in the cytosol (e.g. Michaud et al., 1998; De Jaeger et al., 1999; Rajabi-Memari et al., 2006; Marusic et al., 2007), alternative destinations, such as the chloroplast, the ER or the apoplast, appear to be more appropriate for most proteins.
Figure 1. Subcellular targeting of recombinant proteins in plant cells. Recombinant proteins bearing an N-terminal signal peptide (SP) in their primary sequence enter the cell secretory pathway via the endoplasmic reticulum (ER), and then travel through the Golgi system to be secreted in the apoplast (default pathway) or directed to the vacuole if a vacuolar sorting determinant (VSD) is present in the protein sequence. Proteins secreted into the ER can also be retained in this compartment by the grafting of an ER retention signal – the KDEL (or HDEL) tetrapeptide motif – at the C-terminus. Proteins with no signal peptide accumulate in the cytosol (default location) or migrate to specific organelles when an appropriate peptide signal is included in the transgene sequence. Peptide signals used recently in transgenic plant platforms include plastid (e.g. chloroplast) transit peptides (PTP), nuclear localization signals (NLS) and the tripeptide peroxisome target sequence serine-lysine-leucine (SKL).
Download figure to PowerPoint
Retention in the ER
Targeting to the cell secretory pathway, in particular, has been proposed to improve the stability and yield of several proteins (Ma et al., 2003; Yoshida et al., 2004; Vitale and Pedrazzini, 2005). In practice, the addition of an N-terminal signal peptide sequence to the protein-encoding transgene triggers a co-translational transfer of the nascent protein to the endomembrane system (Chrispeels and Faye, 1996) (Figure 1). Proteins bearing a signal peptide for cellular secretion first enter the ER via the ER protein translocation channel (Galili et al., 1998), and then migrate through this compartment and the Golgi apparatus until reaching the extracellular medium (default pathway) or the vacuole, if a vacuolar sorting signal is found in the primary sequence. Recombinant proteins entering the ER may also be retained in this compartment by simple apposition of the tetrapeptide ER retention signal (K/H)DEL (Michaud et al., 1998) or grafting of a γ-zein proline-rich domain (Mainieri et al., 2004) at the C-terminus. The (K/H)DEL motif is a common ER retrieval signal in eukaryotes, believed to redirect tagged proteins to the ER after their recognition by a (K/H)DEL receptor complex in the Golgi apparatus (Lee et al., 1993; Pagny et al., 2000). In contrast with most reticuloplasmins, cereal prolamins accumulate in the ER of seed cells without the involvement of the (K/H)DEL motif, forming high-density protein bodies by a sequence-specific sorting mechanism yet to be elucidated (Vitale and Pedrazzini, 2005). In practice, the retention of zein (and other prolamins) in the ER, and the concomitant formation of protein bodies, may be easily reproduced in both storage and vegetative tissues of transgenic plants (Geli et al., 1994; Coleman et al., 1996), which makes this system potentially useful for the high-level accumulation of recombinant proteins in planta and their easy recovery in crude extract preparations by gradient centrifugation procedures (Torrent et al., 2006a, b).
Numerous studies have been published illustrating the positive impact of retaining clinically or industrially useful proteins in the ER compartment of plant cells, using, in most cases, a (K/H)DEL retention signal (Ma et al., 2003). The ER, which constitutes a natural reservoir for some storage proteins in seed cells (Shewry and Halford, 2002), can physically accommodate high levels of recombinant protein product in planta (Wandelt et al., 1992). At the biochemical level, the low abundance of proteolytic enzymes and the presence of molecular chaperones in the ER, together with an oxidizing status favouring disulphide bond formation, make this organelle a suitable destination for several proteins susceptible to rapid turnover or showing a complex folding pathway (Nuttall et al., 2002; Faye et al., 2005). Several reports have documented the positive impact of ER retention on the production of recombinant antibodies in terms of protein stability, quality or yield (Schouten et al., 1996; Conrad and Fiedler, 1998; Stoger et al., 2002; Gomord et al., 2004). Similar tendencies have been observed for several other proteins of medical or industrial interest, including, as recent examples, human interleukin-4 (Ma et al., 2005b), the SARS coronavirus S protein antigen (Pogrebnyak et al., 2005), the synthetic silk-like protein DR1B (Yang et al., 2005) and a recombinant phytase from Aspergillus niger (Peng et al., 2006).
Despite these promising developments, the ER cannot be considered as a suitable destination for all proteins. To be stable or active, a number of clinically useful proteins require late post-translational modifications, such as the formation of complex glycans, the addition of a lipid moiety or the proteolytic removal of a propeptide sequence, which may occur downstream of the ER along the secretory pathway, notably in the Golgi, vacuole or apoplast (Gomord and Faye, 2004; Faye et al., 2005). Other proteins may exhibit an altered integrity or structural heterogeneity in the ER, as a result of unintended proteolytic processing by ER-resident proteases (Faye et al., 2005). The ER lumen is generally considered to be a mild environment for labile, e.g. immature, proteins, but proteolytic processing events altering the structure of secreted proteins have been observed in this compartment (Bass et al., 2000; Schmitz and Herzog, 2004). For instance, the bovine plasma protein aprotinin expressed in leaves of transgenic potato showed structural heterogeneity when accumulated in the ER, presumably as a result of the sequential removal of specific amino acids at the N- and C-termini by endogenous peptidases (Badri et al., 2005). A potential solution to prevent unintended proteolytic processing in the ER, or later along the secretory pathway (Sharp and Doran, 2001), is to identify and mutate susceptible amino acid sites by site-directed mutagenesis, as suggested by Outchkourov et al. (2003) for the sea anemone protein equistatin, processed to several truncated forms in the secretory pathway of transgenic potato leaf cells by arginine/lysine- and asparagine-specific cysteine proteases.
Targeting to the apoplast
Simply targeting the protein to an alternative compartment, even downstream in the cell secretory pathway, may also help to prevent unintended processing. For instance, recombinant bovine aprotinin targeted to the apoplast of potato leaf cells can be detected as a homogeneous, unprocessed form, similar in size to the native protein purified from bovine pancreas (Badri et al., 2008), in contrast with the proteolysis-related heterogeneity mentioned above for the same protein retained in the ER. The extracellular medium typically exhibits a high proteolytic content (Callis, 1995; Hellwig et al., 2004; Schiermeyer et al., 2005), but several recombinant proteins tagged for extracellular secretion have been produced successfully in plant or plant cell platforms over the last few years (Gaume et al., 2003; Sojikul et al., 2003; Streatfield et al., 2003; Wirth et al., 2004; Hellwig et al., 2004; Komarnytsky et al., 2004; Gils et al., 2005; Yang et al., 2005). Proteolytic processing in the extracellular medium may even represent an advantage for the correct maturation of certain proteins, as illustrated recently with the dust mite allergenic protein Der p 1, processed to its mature, active form in the culture medium of transgenic BY-2 tobacco cells naturally secreting proteases with hydrolytic activity against the propeptide sequence of this protein (Lienard et al., 2007).
Targeting to the vacuole
Similar to the apoplastic medium, the vacuole represents a suitable accumulation site for several recombinant proteins, especially for production platforms relying on seed tissues (Stoger et al., 2005). The vacuole plays several important roles in planta, including the control of cell turgor, the turnover of macromolecules, the sequestration of toxic secondary metabolites and the storage of high-energy compounds in seeds or vegetative storage tissues (Marty, 1999). Recent evidence in the literature has suggested the occurrence of two distinct types of vacuole in plant cells: lytic (or vegetative) vacuoles, which present an acidic environment rich in hydrolytic enzymes; and protein storage vacuoles, which show a slightly acidic or neutral pH well adapted to protein storage (Robinson et al., 2005). Targeting to the vacuole, although not yet fully understood, is determined by small stretches of amino acids within the protein primary sequence acting as sorting signals to direct the maturing protein towards the vacuole (Neuhaus and Rogers, 1998; Mackenzie, 2005; Vitale and Hinz, 2005). In general, lytic vacuoles are not considered as a suitable destination for recombinant proteins in planta, owing to their high proteolytic content (Goulet and Michaud, 2006). By contrast, protein storage vacuoles present a milder environment compatible with protein accumulation (Stoger et al., 2005), especially in seeds, where they are most abundant (Müntz, 1998; Park et al., 2004).
Good accumulation levels have been reported for a number of recombinant proteins targeted to the vacuole (Table 1), including, for instance, the synthetic analogue of spider dragline silk protein DP1B (Yang et al., 2005), the heat-labile enterotoxin B from E. coli (Streatfield et al., 2003), the toxic biotin-binding proteins avidin and streptavidin (Murray et al., 2002) and a thermostable β-glucanase of bacterial origin (Horvath et al., 2000). In practice, correct in situ localization of recombinant proteins bearing a sorting sequence for vacuolar targeting should be undertaken on a systematic basis, considering the species- or tissue-dependent functionality of some sorting signals (Vitale and Hinz, 2005). A good example of this phenomenon has been provided for a fungal phytase expressed in rice, which was readily detected in the apoplastic environment of leaf tissues, but retained in ER protein bodies and protein storage vacuoles in the seed endosperm (Drakakaki et al., 2006). As illustrated with the silk-like protein DP1B expressed in Arabidopsis (Yang et al., 2005), the impact of vacuolar targeting on the stability and yield of recombinant proteins is also tissue dependent. Although this protein was found at levels reaching 8% of TSP in seed storage vacuoles, no detectable levels of the same protein could be observed in leaf cell vacuoles. In a similar manner, targeting DP1B to the apoplast provided good yields in leaves, but poor yields in seeds (Yang et al., 2005), again stressing the need for an empirical case-by-case assessment of different tissue and cellular destinations for each protein expressed.
Targeting to the chloroplast
The chloroplast, peroxisome and nucleus have been proposed as other cellular destinations for protein production in plant platforms (Daniell et al., 2002; Hyunjong et al., 2006). In practice, recombinant proteins may be sent to these organelles by the inclusion of an appropriate targeting peptide (or localization signal) in the transgene sequence (Figure 1). For instance, the heat-labile toxin Lt-B from enterotoxicogenic E. coli exhibited increased levels in corn grains when diverted from the cytosol to the nucleus by the addition of a nuclear localization signal from the simian virus 40 large T-antigen in the C-terminal position (Streatfield et al., 2003). In a similar manner, a fungal xylanase useful in environment-related technologies showed high accumulation levels in Arabidopsis leaf tissues when sent to chloroplasts using the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase transit peptide, or sent to peroxisomes using the tripeptide targeting motif SKL (serine-lysine-leucine) grafted at the C-terminus (Hyunjong et al., 2006). Another example was the improved production of human growth hormone in N. benthamiana leaf cells when transferred from the cytosol to the chloroplast using the chloroplast transit peptide of rubisco small subunit (Gils et al., 2005).
Efficient procedures have also been devised to insert transgenes in the chloroplast genome, and then to regenerate transplastomic plant lines accumulating high levels of recombinant protein directly in the chloroplast stroma (Daniell et al., 2001; Maliga, 2002). Chloroplast transformation offers several advantages over nuclear transformation, including uniform transgene expression rates, multiple copies of the transgene in each cell, co-expression of multiple genes from the same construct, minimal gene silencing and minimal transgene escape in the environment owing to the maternal inheritance of chloroplast DNA in several species (Daniell et al., 2002). The chloroplast stroma supports protein post-translational modifications, such as multimerization and disulphide bridge formation (Daniell, 2006), making it a suitable environment for the expression of proteins not relying on complex modifications, such as glycosylation, typical of the cell secretory pathway. Several transplastomic plant lines have been engineered over the last 10 years for recombinant protein expression, providing very high yields for a number of useful proteins of prokaryotic and eukaryotic origin, including somatotropin, serum albumin, anthrax protective antigen, cholera toxin B subunit and tetanus toxin fragment C (Daniell et al., 2001, 2005; Tregoning et al., 2003).
However, a number of endogenous proteases are present in the chloroplast (Adam and Clarke, 2002), which can impair the overall stability and accumulation of recombinant proteins. An interesting example has been provided for the rotavirus VP6 protein, which showed high accumulation rates in chloroplasts of young tobacco leaves, but negligible rates in older leaves, despite comparable levels of mRNA transcripts (Birch-Machin et al., 2004). A similar decline in older tissues was observed for a fungal xylanase (Hyunjong et al., 2006) and for the insecticidal Bt toxin Cry2Aa2 (De Cosa et al., 2001), again stressing the importance of a careful assessment of the stability of each recombinant protein in different physiological or environmental contexts. Recombinant protein degradation by chloroplast proteases might appear, however, to be a non-relevant issue in terms of net production yields for some proteins expressed at very high levels (Daniell, 2006). The proteolysis-labile protein human serum albumin, for instance, was found at levels reaching 11% of TSP in transplastomic tobacco lines developed using chloroplast untranslated regions in gene constructs, in sharp contrast with levels below 0.02% of TSP in lines developed using the commonly employed Shine–Delgarno regulatory sequence (Fernandez-San Millan et al., 2003). This dramatic increase, probably the result of an increased expression rate of the transgene, was also associated with the formation of large inclusion bodies in vivo (Fernandez-San Millan et al., 2003), which presumably sequestered the recombinant protein and prevented its hydrolysis in planta, as described earlier for a number of proteins expressed in heterologous environments (Enfors, 1992).