WO2012168794A1

WO2012168794A1 - Affinity trapping agent and uses thereof

Info

Publication number: WO2012168794A1
Application number: PCT/IB2012/001268
Authority: WO
Inventors: Véronique GOMORD; Loïc FAYE; Marc-André D'Aoust; Manon Couture; Sonia Trepanier; Louis-Philippe Vezina; Anne Catherine FITCHETTE
Original assignee: Angany Genetics; Medicago, Inc.
Priority date: 2011-06-07
Filing date: 2012-06-06
Publication date: 2012-12-13

Abstract

A nucleic acid encoding an affinity trapping agent is provided. The affinity trapping agent comprises a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal. Methods for targeting the immobilization of a recombinant peptide or protein of interest, or an endogenous peptide or protein of interest, to a subcellular compartment of a cell are also provided. If the peptide or protein of interest is recombinant then a nucleotide sequence encoding the recombinant peptide or protein of interest and a nucleic acid encoding the affinity trapping agent are introduced into the cell and co- expressed to form a complex thereby targeting the immobilization of the recombinant peptide or protein of interest. If the peptide or protein of interest is endogenous, then the nucleic acid encoding the affinity trapping agent having a binding affinity for the endogenous peptide or protein of interest is introduced into the cell, and the nucleic acid encoding the affinity trapping agent is expressed in the cell to form a complex thereby targeting immobilization of the endogenous peptide or protein of interest.

Description

Affinity trapping agent and uses thereof

FIELD OF INVENTION

[0001] The present invention relates to affinity trapping agents and methods of using these affinity trapping agents. BACKGROUND OF THE INVENTION

[0002] A main objective in plant molecular farming is to improve the quantity and quality of plant-made pharmaceuticals (PMPs). The high levels of expression recently obtained using either transient expression in agro-infiltrated leaves (Marcello et al,

2009, Vezina et al, 2009, D'Aoust et al. 2010) or viral-assisted expression systems (Giritch et al. 2006, Pogue et al. 2010) have illustrated productivity and rapidity for the production of complex therapeutic targets in plant expression systems.

[0003] Protein targeting to subcellular compartments using appropriate sorting peptides is one strategy that may be used to improve PMP's yield and quality. For example, targeting of recombinant proteins in the plant endoplasmic reticulum (ER) or in protein body-like organelles after fusion with H/KDEL, prolamin, elastin-like peptides or hydrophobin tags at their terminal ends have resulted in increased yield and prevention of plant- specific modifications on N-glycans (Gomord et al., 2004. Conley et al,.2010). Targeted-expression of maturation enzymes in different subcellular compartments has also resulted in higher biological activity of PMPs, and/or increased in vivo stability or reduced immunogenicity. Examples of humanization of N-glycosylation in plant expression systems have been previously described (Frey et al., 2009, Vezina et al, 2009, Castilho et al, 2010, Loos et al.,

2010, Gomord et al, 2010).

[0004] Targeting of either PMPs or posttranslational maturation enzymes to the different compartments of the plant secretory pathway has required fusion of the protein of interest with specific targeting signals (Saint-Jore-Dupas et al., 2007). Targeting of recombinant proteins to particular intracellular compartments within a plant using peptidic targeting signals fused to the recombinant protein backbone results in increased yield and quality (see Benchabane et al, 2009 for a recent illustration). Production of recombinant antibodies fused to a H/KDEL retention signal, increase protein stability, quality and yield (Schouten et al, 1996; Conrad and Fiedler, 1998; Stoger et al, 2002; Gomord et al, 2004, Petrucelli et al, 2006; Floss et al, 2008). However, this approach has two major drawbacks. The first being that for each protein and for each subcellular location studied, a new fusion has to be prepared and expressed (e.g. see Conley et al, 2008, Benchabane et al, 2009). Similarly, glycoengineering of plant expression systems using targeted expression of heterologous glycosyltransferases (GT) in the plant secretory pathway involved preparing and expressing different chimeric forms of each GT targeted to different compartments of the secretory pathway (Vezina et al, 2009, Frey et al, 2009, Saint- Jore-Dupas et al, 2007). The second drawback being that proteolytic events at a C- or N-terminus of the protein of interest may cleave retrieval or targeting sequences fused to the protein. For example, cleavage of the KDEL tag as a result of protease susceptible sites at the C-terminus of bovine recombinant aprotinin has been reported (Badri et al, 2009). Problems may arise due the presence of modified signal sequences on a protein of interest. Additionally, tags sequences are not desired on pharmaceutical proteins, as non-native proteins with added domains are a concern from the safety perspective.

[0005] The ability of antibodies to bind antigens with high specificity and affinity makes them useful for the prevention, diagnosis and treatment of diseases in animals, including human diseases. This use has increased dramatically with the advent of recombinant antibody technology and the possibility of using cultured mammalian cells for production of antibody- based drugs. Antibody expression in transgenic plants is known (Hiatt, 1989), including production of full length antibodies and antibody fragments in several plant species (see Faye and Gomord, 2010). The use of plant-derived antibodies ex planta as diagnostic tools, for immunochromatography or in medical therapy has also been described (Gomord et al, 2004; Schirrman et al, 2008; Ko et al, 2009; De Muynck et al, 2010 for recent reviews).

[0006] The diversity and specificity of antibodies expressed in vivo is also useful for immunomodulation where the antibody or antibody fragment is used to affect an intracellular target antigen. Immunomodulation was originally developed in mammalian cells where the binding of an antibody to an intracellular target molecule was used to block, suppress, alter or even enhance the process mediated by this antigen molecule.

[0007] The use of intracellular antibody-mediated in vivo targeting, or intrabody- mediated in vivo targeting, for therapeutic applications in humans was developed as an alternative to gene knock out in mammalian cells (Pumphrey and Marasco, 1998,

Deshane et al, 1995; Richardson et al, 1995; Stocks, 2004). The antibodies or antibody fragments remain within the cell membrane to prevent secretion and/or maturation of a target protein that would normally be expressed on the cell surface. ScFv intrabodies targeted to the ER have been used as potential therapeutic agents to inactivate cell surface receptors and oncoproteins or to inhibit virus replication in mammalian cells .

[0008] Immunomodulation in plants has been used to change agronomic traits or to protect plants against pathogen infection (see De Jaeger et al., 2000, Conrad and Manteuffel, 2001 for reviews). Immunomodulation of ABA functions in different plant organs and tissues revealed additional information of the role of this

phytohormone in seed development, during early embryogenesis and in stomata development (Artsaenko et al., 1995, Phillips et al., 1997, Senger et al., 2001; Wigger et al., 2002). The most common antibody format used for in planta

immunomodulation involves the scFv as a fusion of the variable regions of the heavy and the light chains of immunoglobulins linked together with a short polypeptide linker. This chimeric molecule retains the specificity of the original immunoglobulin is of a smaller size and exhibits lower susceptibility to proteolysis. In addition scFv fragments circumvent post-translational modifications required for full size antibody activity and stability, such as assembly of heavy and light chains, formation of intra chain disulfide bonds and glycosylation. Expression of a scFv in plant cell were either soluble scFvs expressed in the cytosol, the apoplasm, the ER, or a membrane bound scFv targeted to the plasma membrane (Schillberg et al., 2000). These scFv intrabodies were used in planta for immunomodulation of physiological functions or to confer pathogen or herbicide resistance (Artsaenko et al., 1995; Eto et al., 2003; Olea-Popelka et al, 2005; Villani et al, 2005; David et al, 2007). SUMMARY OF THE INVENTION

[0009] The present invention relates to affinity trapping agents and methods of using these affinity trapping agents.

[0010] It is an object of the invention to provide an improved affinity trapping agent.

[0011] According to the present invention there is provided a nucleic acid encoding an affinity trapping agent, the affinity trapping agent comprising a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal. The peptide or protein ligand may be selected from the group: single chain variable fragment (scFv), Protein A, Protein L, Protein G, Protein A/G. Furthermore, the heterologous subcellular compartment targeting signal targets the affinity trapping agent to a subcellular compartment selected from the group: endoplasmic reticulum, Golgi network, chloroplast, cytosol, mitochondria, nucleus and peroxisome. The Golgi network may further comprise cis- Golgi, median-Golgi, and trans-Golgi.

[0012] The present invention also provides a method (A) for targeting the

immobilization of a recombinant peptide or protein of interest to a subcellular compartment of a cell comprising, i) introducing a nucleotide sequence encoding the recombinant peptide or protein of interest into the cell; ii) introducing a nucleic acid encoding an affinity trapping agent into the cell, the affinity trapping agent comprising a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal; and iii) co-expressing the nucleotide sequence encoding the recombinant peptide or protein of interest and the nucleic acid encoding the affinity trapping agent in the cell to form a complex and targeting the immobilization of the recombinant peptide or protein of interest. [0013] The present invention also provides the method (A) as just described wherein the recombinant peptide or protein of interest may be either soluble or membrane bound. Furthermore, the recombinant peptide or protein of interest may be soluble, and the affinity trapping agent membrane bound, the recombinant peptide or protein of interest may be membrane bound, and the affinity trapping agent membrane bound, the recombinant peptide or protein of interest may be membrane bound, and the affinity trapping agent soluble and comprises a subcellular compartment targeting signal; or the recombinant peptide or protein of interest may be soluble, and the affinity trapping agent soluble and comprises a subcellular compartment targeting signal. The nucleotide sequence encoding the recombinant peptide or protein of interest may be stably or transiently expressed. The nucleic acid encoding the affinity trapping agent can be stably or transiently expressed. The recombinant peptide or protein of interest may be a plant made pharmaceutical, an enzyme, or a protein maturation enzyme. The recombinant peptide of interest may comprise a FLAG tag epitope. Alternatively, the affinity trapping agent may comprise an affinity tag, for example, a FLAG tag epitope, poly (His), Strep Π, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag, and the epitope or tag is used to purify the complex.

[0014] The present invention also pertains to the method (A) as described above wherein: a) in the step of introducing, step i), the nucleotide sequence is introduced into the cell in a transient manner; b) in the step of introducing, step i), the nucleic acid sequence is introduced into the cell in a transient manner; or c) in the steps of introducing, step i) and step ii), the nucleotide sequence and the nucleic acid sequence are introduced into the cell in a transient manner.

[0015] Furthermore, in the method (A) as described above, the cell may be a plant cell. For example the plant cell may be a Nicotian benthamiana cell, a Nicotiana tabaccum cell, a Medicago sativa cell, or an Arabidopsis thaliana cell. [0016] The present invention also provides a method (B) for targeting the immobilization of an endogenous peptide or protein of interest to a subcellular compartment of a cell comprising, i) introducing a nucleic acid encoding an affinity trapping agent into the cell, the affinity trapping agent comprising a peptide or protein ligand having a binding affinity for the endogenous protein of interest, fused to a heterologous subcellular compartment targeting signal; and ii) expressing the nucleic acid encoding the affinity trapping agent in the cell to form a complex and targeting the immobilization of the endogenous peptide or protein of interest.

[0017] The endogenous peptide or protein of interest may be either soluble or membrane bound. The endogenous peptide or protein of interest may be an enzyme endogenously expressed by the cell. The affinity trapping agent may comprise a FLAG tag epitope, a FLAG tag epitope, poly (His), Strep Π, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag, and the epitope or tag is used to purify the complex..

[0018] The present invention provides the method (B) as just described, wherein i) the endogenous peptide or protein of interest is soluble, and the affinity trapping agent is membrane bound; ii) the endogenous peptide or protein of interest is membrane bound, and the affinity trapping agent is membrane bound iii) the endogenous peptide or protein of interest is membrane bound, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal; or iv) the endogenous peptide or protein of interest is soluble, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal. [0019] The present invention also pertains to the method (B) described above wherein in the step of introducing, step i), the nucleic acid sequence is introduced into the cell in a transient manner.

[0020] The present invention includes the method (B) described above, wherein the cell is a plant cell. The plant cell may be a Nicotian benthamiana cell, a Nicotiana tabaccum cell, a Medicago sativa cell, or an Arabidopsis thaliana cell.

[0021] The present application describes an approach based on affinity trapping that allows targeting of a recombinant or endogenous peptide or protein of interest in any subcellular compartment. An advantage of the strategy described herein is that there is no need for a modification of the peptide or protein of interest. Targeting of the peptide or protein of interest is mediated through the binding of the protein of interest to the affinity trapping agent, and not necessarily on localization signals fused to the protein of interest. An epitope tag may be added to the protein of interest or the affinity trapping agent, to assist in purification.

[0022] Current attempts at improving the quantity or quality of a biopharmaceutical protein produced in plant expression systems are often based on targeted expression of either the biopharmaceutical itself or the enzymes responsible of key events in its posttranslational maturations. Current approaches are based on the expression of the protein of interest in different sub-cellular compartments, these are time consuming and provide ambiguous results if different chimeric forms of a same protein are obtained in different cellular locations and compared.

[0023] The present invention provides targeted expression of a peptide or protein using affinity trapping. The peptide or protein of interest and an affinity trapping agent comprising binding affinity for the peptide or protein of interest and fused to chosen targeting signals may be either both stably or transiently co-expressed, or with one being stably expressed with the other being transiently expressed. As

demonstrated herein, any combination of soluble or membrane bound affinity trapping agent together with a soluble or membrane bound peptide or protein of interest is suitable for in vivo targeting of the peptide or protein of interest into the endoplasmic reticulum, or Golgi subcompartments. [0024] The affinity trapping agent-mediated in vivo targeting strategy provides a general tool for a fast identification of a suitable cellular compartment for production of a protein, for example a biopharmaceutical protein, enzyme, or maturation enzyme. The methods described herein do not require the use of tags on the peptide or protein of interest, and makes the methods suitable for plant made pharmaceutical production.

[0025] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0027] FIGURE 1 shows a schematic representation of constructs described herein. Soluble ER scFv (SolERscFv) is a soluble antibody fragment directed against green fluorescent protein (GFP - the anti-GFP portion comprising Vh-(G₄S)3-VI) fused to the signal peptide of tobacco chitinase (SP) and to a C-terminal ER localisation tetrapeptide HDEL via a FLAG tag and a (His)6 sequence; Membrane ER scFv is a membrane-bound antibody fragment directed against GFP fused to the N-terminal 90 amino acids of Arabidopsis thaliana glucosidase I (GCS90; comprising a cytoplasmic tail-transmembrane domain-and a stem) for retention in the ER membrane

(previously disclosed in Boulaflous et al., 2010; which is incorporated herein my reference); Membrane Golgi scFv is a membrane-bound antibody fragment directed against GFP fused to the N-terminal 99 first amino acids of Glycine max mannosidase I (Man99; comprising a cytoplasmic tail-transmembrane domain-and a stem) for retention in the czs-Golgi (previously disclosed in Saint -Jore-Dupas et al., 2006; which is incorporated herein by reference); SP-eCFP is an enhanced cyan fluorescent protein (eCFP) variant fused to the signal peptide of tobacco chitinase; sGFP-Der p 2Y is a Der p 2Y glycoallergen secreted in the extracellular space fused to GFP sGFP-Der p 2Y-HDEL is the Der p 2Y glycoallergen fused to GFP and to the C- terminal ER localisation tetrapeptide HDEL (previously disclosed in Boulaflous et al.,2009; which is incorporated herein by reference); mRFP-HDEL is a monomeric red florescent protein (mRFP) fused to the N-terminal signal peptide (SP) of tobacco chitinase and the C-terminal ER localisation tetrapeptide HDEL (previously disclosed in Boulaflous et al.,2009; which is incorporated herein by reference); Cis Golgi marker comprises Man99 (see Saint-Jore-Dupas et al., 2006; which is incorporated herein by reference) fused to either CFP or GFP; Medial Golgi marker comprises XylT35 (see Saint-Jore-Dupas et al., 2006; which is incorporated herein by reference) fused to either mRFP, GFP or CFP ; Trans Golgi marker comprises ST52 (see Boulaflous et al., 2009; which is incorporated herein by reference) fused to mRFP or GFP, CFP ; (G₄S)3 : linker comprising 3 repetitions of (Gly₄Ser); Vh : variable heavy chain; VI : variable light chain; TagHDEL : Flag(His)6-HDEL; CT : cytosolic tail; TMD : transmembrane domain; S : stem.

[0028] Figure 2 shows expression of green florescent protein (GFP)- specific scFvs (Soluble ER scFv as described in Figure 1) in leaf epidermal tobacco cells. Three days after syringe agroinfiltration used for transient expression, protein extracts from tobacco leaves agroinfiltrated with either an empty vector (lane 1), or with vectors used for expression of scFvl (lane 2), scFv2 (lane 3), scFv3 (lane 4) were separated in a 12% SDS-PAGE gel and either staining with Coomassie blue (panel A) or transferred on nitrocellulose for western blot analysis using a monoclonal anti-Flag antibody (panel B).

[0029] Figure 3 shows that that SolER-scFv relocalizes Golgi anchored proteins to the ER, and that SolER-scFv is specific for green florescent protein (GFP) and its spectral variants, cyan florescent protein (CFP) and yellow florescent protein (YFP), but does not react with monomelic red fluorescent protein (mRFP). All epidermal cells presented in this figure co-express at least two Golgi membrane proteins. Panel A shows co-expression of XYLT35 fused to CFP and XYLT35 fused to mRFP; Panel B shows co-expression of XYLT35 fused to CFP, XYLT35 fused to mRFP, and sol ER- scFv; Panel C shows co-expression of XYLT35 fused to GFP and XYLT35 fused to mRFP; Panel D shows co-expression of XYLT35 fused to GFP, XYLT35 fused to mRFP, and sol ER-scFv; Panel E shows co-expression of XYLT35 fused to YFP and XYLT35 fused to CFP; Panel F shows co-expression of XYLT35 fused to YFP, XYLT35 fused to CFP, and sol ER-scFv. The Golgi markers co-localize in the Golgi (A, C, E; fluorescence shows as spots). When Golgi markers are co-expressed with anti-GFP (SolER-scFv), GFP and its spectral variants, CYP or RFP are relocalized to the ER (B, D, F; shows as diffuse fluorescence within the ER), but mRFP stays into the Golgi (B, D; fluorescence shows as spots). Scale bars = 8 μηι.

[0030] Figure 4 shows that SolER-scFv relocalizes secreted soluble proteins to the ER as determined using confocal laser scanning microscopy (CLSM) analysis of N.

benthamiana leaf epidermal cells. Panel A shows expression of SP-eCFP; Panel B shows expression of mRFP-HDEL; Panel C shows the results of Panel A and B merged together; Panel D shows co-expression of SP-eCFP and solER-scFv; Panel E shows co-expression of mRFP-HDEL and solER-scFv; Panel F shows Panel D and E merged together; Panel G shows expression of SP-eCFP; Panel H shows expression of ST52-mRFP; Panel I shows Panel G and H merged together; Panel J shows co- expression of SP-eCFP and solER-scFv; Panel K shows co-expression of ST52-mRFP and solER-scFv; Panel I shows Panel J and K merged together. SP-eCFP is exclusively secreted in the extracellular space (Panels A-C and G-I; shows as a band of fluorescence along outer edge of cell), while SP-eCPF is retained in the ER when co expressed with the SolER-scFv (see Panels D-F and J-L; shows as diffuse fluorescence within ER). As noted in Figure 3, the subcellular localization of mRFP trans-Golgi marker is not affected in presence of the solER-scFv (see panels H and ; fluorescence shows a spots). Bars = 8 μιη.

[0031] Figure 5 shows that solER-scFv relocalizes membrane proteins to the ER as determined using confocal laser scanning microscopy (CLSM) analysis of Nicotiana tabacum leaf epidermal cells. Panel A shows co-expression of Man99-GFP and ST52-mRFP; Panel B shows co-expression of Man99-GFP, ST52-mRFP, and solER- scFv; Panel C shows co-expression of Man99-GFP and Man99-mRFP; Panel D shows co-expression of Man99-GFP, Man99-mRFP, and solER-scFv; Panel E shows co- expression of XYLT35-GFP and XYLT35-mRFP; Panel F shows co-expression of

XYLT35-GFP, XYLT35-mRFP and solER-scFv; Panel G shows co-expression of M99-TMD23-GFP (Saint- Jore-Dupas et al., 2006, which is incorporated herein by reference) and XYLT35-mRFP; Panel H shows co-expression of M99-TMD23-GFP [], XYLT35-mRFP, and solER-scFv. GFP fusion Golgi markers are relocalized to the ER in presence of the SolER-scFv (see Panels B, D, F, H; shows as diffuse fluorescence within the ER) while the location of the mRFP marker in the medial Golgi is not affected by this antibody fragment (F, H; fluorescence shows as spots). Bars = 8 μιη.

[0032] Figure 6shows that Golgi membrane scFv traps secreted soluble proteins into the Golgi as determined using confocal laser scanning microscopy (CLSM) analysis of Nicotiana tabacum leaf epidermal cells. Panel A shows expression of SP-eCFP;

Panel B shows expression of mRFP-HDEL; Panel C shows Panel A and B merged together; Panel D shows co-expression of SP-eCFP and membrane Golgi-scFv; Panel E shows co-expression of mRFP-HDEL and membrane Golgi-scFv (mb Golgi scFv); Panel F shows Panel D and E merged together; Pane G shows co-expression of SP- eCFP and membrane Golgi-scFv; Panel H shows co-expression of ST52-mRFP (trans

Golgi Marker) and membrane Golgi-scFv; Panel I shows Panel G and H merged together. The secreted SP-eCFP (see A, C; expression shows as a band of fluorescence around an outer edge of cell) is relocated in the Golgi in presence of the membrane Golgi scFv (D, F, G, I; shows as spot of fluorescence) where it co-localizes with the Golgi marker (I; shows as spots of fluorescence). Bars = 8 μπι.

[0033] Figure 7 shows that Golgi membrane scFv relocalizes membrane proteins into the Golgi. Panel A shows expression of cis Golgi marker (Man99-eCFP; early Golgi CFP) in epidermal cells; Panel B shows expression of medial Golgi marker (XYLT35- mRFP); Panel C shows Panel A and B merged together. Panel D shows co-expression of cis Golgi marker (Man99-eCFP; early Golgi CFP) and membrane ER-scFv (ERmb- scFv); Panel E shows co-expression of medial Golgi marker (XYLT35-mRFP) and membrane ER-scFv; Panel F shows Panel D and E merged together; Panel G shows expression of R/LGCS90-GFP (described in Boulaflous et al, 2010, which is incorporated herein my reference); Panel H shows expression of medial Golgi marker (XYLT35-mRFP); Panel I shows Panel G and H merged together; Panel J shows co- expression of R/LGCS90-GFP and membrane Golgi -scFv; Panel K shows co- expression of medial Golgi marker (XYLT35-mRFP) and membrane Golgi -scFv; Panel L shows Panel J and K merged together. Epidermal cells expressing Golgi membrane proteins fused to mRFP (B,E) or to CFP (A,D) localize to the Golgi (shown as spots of fluorescence in Panels A, B, D and E). When ER membrane scFv is added, CFP is immunotrapped in the ER (D; shows as diffuse florescence within ER), whereas mRFP remains in the Golgi (E,F; shows as spots of fluorescence). Epidermal cells expressing Golgi membrane proteins fused to mRFP (H, K) and GFP (G, J) localize to the Golgi (shown as spots of fluorescence in Panels G, H, J, K), When Golgi membrane scFv is added, GFP is immunotrapped in the early Golgi and faintly in the ER (J) and only partially overlaps with the trans-Golgi marker (L). Compare the inserts on micrographs I and L (enlarged three times) to clearly see the

GFP membrane fusion was moved from the trans Golgi to the early Golgi by the mb Golgi scFv. Bars= 8 μπι.

[0034] Figure 8 shows that soluble or membrane ER-scFv relocalizes a secreted biopharmaceutical into the ER, as determined using confocal laser scanning microscopy (CLSM) analysis of Nicotiana tabacum leaf epidermal cells. Panel A shows expression of sGFP-Derp2Y; Panel B shows expression of mRFP-HDEL; Panel C shows Panel A and B merged together; Panel D shows co-expression of sGFP-Derp2Y and solER-scFV; Panel E shows co-expression of mFRP-HDEL and solER-scFV; Panel F shows Panel D and E merged together; Panel G shows co- expression of sGFP-Derp2Y and membrane Golgi-scFv; Panel H shows co- expression of trans Golgi marker (ST52-mRFP) and membrane Golgi-scFv; Panel I shows Panel G and H merged together. The sGFP-Derp2Y glycoallergen is relocated either into the ER when co-expressed with the SolER-scFv (D-F; shows as diffuse florescence within the ER), or into the Golgi apparatus when co-expressed with the mb Golgi scFv, respectively (G-I; shows as spots of fluroescence). Bars = 8 μιη.

[0035] Figure 9 shows N-glycosylation of the Derp2Y glycoallergen is retained into the ER by interaction with the SolER-scFv. (A) Derp2, and a neoglycosylated form of this allergen Derp2Y, were expressed in tobacco cells and purified by immobilized metal affinity chromatography (IMAC affinity chromatography). Purified Derp2 and Derp2Y were then analyzed by SDS-PAGE (proteins) and affino- or immuno-blotting with glycan- specific probes. Derp2Y was immunodetected with antibodies specific for Derp2 (Allergens), for the al,3 fucose and the β1,2 xylose (al,3Fuc/pi,2 xyl) or for the Lewis a (Lewis a) glycoepitopes and affinodetected with the ConA lectin specific for the High-mannose type N-glycans (High-man). In contrast, Derp2, which is not glycosylated, reacts exclusively with antibodies specific for the allergen. (B) Protein extracts from tobacco leaf epidermal cells expressing GFP-Derp2Y fused to the HDEL ER retention signal either alone (lanes 1-2) or with SolER-scFv (lanes 3-4) were resolved by 12% (w/v) SDS-PAGE and immunodetected with anti-Derp2 antibodies via their transfer onto a nitrocellulose membrane. The endoH analysis shows that GFP-Derp2Y co-expressed with SolERscFv exclusively harbours high mannose type N-glycans as it is observed for the GFP-Derp2-HDEL. [0036] Figure 10 shows relocalization of therapeutical glycoproteins in the ER or in the Golgi in different plant systems, as determined using confocal laser scanning microscopy (CLSM) analysis of various plant systems. Panel A shows co-expression of sGFP-Derp2Y glycoallergen and solER-scFv in Medicago sativa; Panel B shows co-expression of sGFP-Derp2Y glycoallergen and solER-scFv in Tobacco BY-2 cells; Panel C shows co-expression of sGFP-Derp2Y glycoallergen and membrane Golgi- scFv in Tobacco BY-2-cells; Panel D shows co-expression of sGFP-Derp2Y glycoallergen and membrane Golgi-scFv in Arabidopsis thalinana. In all cases, the secreted biopharmaceutical is relocalized in a scFv-dependent manner and accumulated accordingly either in the ER of Medicago sativa leaf epidermal cells (A; shows as diffuse fluorescence in the ER), and tobacco BY2 cells (B; shows as diffuse fluorescence in the ER) when it is co-expressed with a SolER-scFv; or in the Golgi of tobacco BY2 cells (C; shows as spots of fluorescence) and Arabidopsis thaliana leaf epidermal cells (D; shows as spots of fluorescence) when co-expressed with a Golgi membrane scFv. Bars = 8 μιη. [0037] Figure 11 shows a schematic representation of combinations used for illustrating scFv-mediated targeting in the present study. A soluble scFv is efficient for in vivo targeting of a soluble (A) or a membrane (B) protein. A membrane bound scFv can interact and relocate its soluble (C) or membrane bound (D) protein target.

DETAILED DESCRIPTION [0038] The present invention relates to affinity trapping agents and methods of using these affinity trapping agents.

[0039] The following description is of a preferred embodiment.

Intrabody -mediated in vivo targeting [0040] The present invention provides a method for targeted accumulation of recombinant proteins in cells, for example but not limited to plant cells. The targeted accumulation of the protein of interest within the cell is dependent on interaction between the protein of interest and a ligand, peptide or protein comprising an affinity binding capacity directed to the protein of interest. The ligand, peptide or protein comprising an affinity binding capacity may be soluble or it may be membrane bound. Examples of the ligand, peptide or protein may include but are not limited to an intracellular scFv, an scFv variant, bivalent scFv, trivalent scFv, Protein A, Protein A/G, Protein G, Protein L, or other peptide or protein that is characterized as having an affinity binding capacity to a specific target.

[0041] An scFv format for antibody- mediated targeting is used herein as an example of a peptide or protein characterized as having an affinity binding capacity to a specific target. The physical linkage of the heavy and light chain variable region of the scFv to facilitates the correct association and interaction of the heavy and light chains, so that the scFv does not require access to accessory proteins for proper folding. Therefore, the approach described herein, using ER- targeted, or Golgi- targeted, scFv fragments, may also be used with scFv fragments that obtain their three dimensional form in the cytoplasmic environment, bind with a target protein, and then shuttle the target protein to different subcellular locations through the use of appropriate cellular localization signals such as localization signals for the chloroplast, cytosol, mitochondria, nucleus or peroxisome.

[0042] The present invention therefore provides a nucleic acid encoding an affinity trapping agent. The affinity trapping agent comprises a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal. The heterologous subcellular compartment targeting signal targets the affinity trapping agent to a subcellular compartment selected from the group: endoplasmic reticulum, Golgi network, chloroplast, cytosol, mitochondria, nucleus and peroxisome. The Golgi network may further comprise cis-Golgi, median- Golgi, and trans-Golgi. Examples of subcellular targeting signals include but are not limited to GCS90 (Boulaflous et al., 2010; which is incorporated herein my reference), Man99 (Saint -Jore-Dupas et al., 2006; which is incorporated herein by reference), XYLT35 (Saint -Jore-Dupas et al., 2006; which is incorporated herein by reference), ST50 (Boulaflous et al., 2009; which is incorporated herein by reference).

[0043] The present invention also includes a nucleotide sequence that hybridizes under stringent hybridisation conditions to a complement of a nucleotide sequence encoding the affinity trapping agent, the subcellular compartment targeting signal, or both the affinity trapping agent and the subcellular compartment targeting signal. Hybridization under stringent hybridization conditions is known in the art (see for example Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3^rd edition 2001 ; each of which is incorporated herein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4 X SSC at 65°C, followed by washing in 0.1 X SSC at 65°C for an hour, or 2 washes in 0.1 X SSC at 65°C each for 20 or 30 minutes.

Alternatively, an exemplary stringent hybridization condition could be overnight (16- 20 hours) in 50% formamide, 4 X SSC at 42°C, followed by washing in 0.1 X SSC at 65°C for an hour, or 2 washes in 0.1 X SSC at 65°C each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaP0₄ buffer pH 7.2; 10 mM EDTA) at 65°C, with 2 washes either at 50°C in 0.1 X SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65°C in 2 X SSC, 0.1% SDS for 20 or 30 minutes each. Additionally, the present invention includes nucleotide sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the nucleotide sequence encoding the affinity trapping agent, the subcellular compartment targeting signal, or both the affinity trapping agent and the subcellular compartment targeting signal.

[0044] Such a sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (using, for example but not limited to, the following parameters: GAP penalty 5, #of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and BLAST, available through the NIH.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement), or using Southern or Northern hybridization under stringent conditions (see Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982). Preferably, sequences that are substantially similar and exhibit at least about 80% and most preferably at least about 90% sequence similarity over a defined length of the molecule, while retaining the activity of the affinity trapping agent, the subcellular compartment targeting signal, or both the affinity trapping agent and the subcellular compartment targeting signal.

[0045] The present invention also provides a method for targeting the immobilization of a recombinant peptide or protein of interest to a subcellular compartment of a cell. This method comprises, introducing a nucleotide sequence encoding the recombinant peptide or protein of interest and a nucleic acid encoding an affinity trapping agent. The affinity trapping agent comprising a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal. The nucleotide sequence encoding the recombinant peptide or protein of interest and the nucleic acid encoding the affinity trapping agent are co-expressed in the cell to form a complex thereby targeting immobilization of the recombinant peptide or protein of interest. The method may involve introducing, the nucleotide sequence, the nucleotide sequence or both into the cell in a stable manner or in a transient manner

[0046] The recombinant peptide or protein of interest may be a cell made pharmaceutical, such as for example a plant made pharmaceutical, an enzyme, or a protein maturation enzyme. The recombinant peptide of interest may comprise a FLAG tag epitope. Alternatively, the affinity trapping agent may comprise a FLAG tag, and the tag is used to purify the complex. As would be known to one of skill in the art (see Lichty J.J., et. al. Prot Exp Purification 41:98- 104, 2005), other affinity tags or epitopes may be used to assist with purification of the complex, for example, but not limited to poly (His), Strep II, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), or epitope tags for example, V5-tag, c- myc-tag, hemagglutinin (HA)-tag.

[0047] An affinity trapping agent comprises a ligand, peptide or protein that is characterized as having an affinity binding capacity directed to a peptide or protein of interest (specific target or coding region of interest). The affinity agent may be soluble or it may be membrane bound. Non-limiting examples of the ligand, peptide or protein may include but are not limited to an intracellular scFv, an scFv variant, bivalent scFv, trivalent scFv, Protein A, Protein A/G, Protein G, Protein L, or other peptide or protein, provided that the ligand, peptide or protein exhibits an affinity binding capacity to a specific target, peptide or protein of interest.

[0048] By nucleotide sequence encoding a "peptide or protein of interest", "specific target" or "coding region of interest" (these terms may be used interchangeably), it is meant any nucleotide sequence, or coding region that is to be expressed as described herein within a plant or portion of the plant. Such a nucleotide sequence of interest may include, but is not limited to, a sequence or coding region whose product is a peptide protein of interest. Examples of a peptide or protein of interest include, for example but not limited to, an industrial enzyme for example, cellulase, xylanase, protease, peroxidase, subtilisin, a protein supplement, a nutraceutical, a value-added product, or a fragment thereof for feed, food, or both feed and food use, a

pharmaceutically active protein, for example but not limited to growth factors, growth regulators, antibodies, antigens, and fragments thereof, or their derivatives useful for immunization or vaccination and the like. Additional peptide or proteins of interest may include, but are not limited to, interleukins, for example one or more than one of IL-1 to IL-24, IL-26 and IL-27, cytokines, Erythropoietin (EPO), insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinations thereof, interferons, for example, interferon- alpha, interferon-beta, interferon- gama, blood clotting factors, for example, Factor VIII, Factor ΓΧ, or tPA hGH, receptors, receptor agonists, antibodies, neuropolypeptides, insulin, vaccines, growth factors for example but not limited to epidermal growth factor, keratinocyte growth factor, transformation growth factor, growth regulators, antigens, autoantigens, fragments thereof, or combinations thereof [0049] The present invention also provides a method for targeting the immobilization of an endogenous peptide or protein of interest to a subcellular compartment of a cell. This method comprises introducing a nucleic acid encoding an affinity trapping agent into the cell. The affinity trapping agent comprising a peptide or protein ligand having a binding affinity for the endogenous protein of interest, fused to a

heterologous subcellular compartment targeting signal. The nucleic acid encoding the affinity trapping agent is expressed in the cell to form a complex thereby targeting the immobilization of the endogenous peptide or protein of interest. The endogenous peptide or protein of interest may be either soluble or membrane bound, for example it may be an enzyme endogenously expressed by the cell. The nucleic acid sequence may be introduced into the cell in a stable manner or in a transient manner.

[0050] The one or more than one or more chimeric genetic constructs of the present invention may be expressed in any suitable plant host that is transformed by the nucleotide sequence, or constructs, or vectors of the present invention. Examples of suitable hosts include, but are not limited to, agricultural crops including alfalfa, canola, Brassica spp., maize, Nicotiana spp., alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton and the like. For example, which is not to be considered limiting, the cell may be a any type of cell, such as for example a plant cell, for example but not limited to a Nicotian benthamiana cell, a Nicotiana tabaccum cell, a Medicago sativa cell, or an Arabidopsis thaliana cell.

[0051] The use of the terms "regulatory region", "regulatory element" or "promoter" in the present application is meant to reflect a portion of nucleic acid typically, but not always, upstream of the protein coding region of a gene, which may be comprised of either DNA or RNA, or both DNA and RNA. When a regulatory region is active, and in operative association, or operatively linked, with a gene of interest, this may result in expression of the gene of interest. A regulatory element may be capable of mediating organ specificity, or controlling developmental or temporal gene activation. A "regulatory region" may includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory elements or transcriptional enhancers. "Regulatory region", as used herein, may also includes elements that are active following transcription, for example, regulatory elements that modulate gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. Several of these latter elements may be located proximal to the coding region. [0052] In the context of this disclosure, the term "regulatory element" or "regulatory region" typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site. However, it is to be understood that other nucleotide sequences, located within introns, or 3' of the sequence may also contribute to the regulation of expression of a coding region of interest. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element. Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence comprised of adenosine and thymidine nucleotide base pairs usually situated approximately 25 base pairs upstream of a transcriptional start site. A promoter element comprises a basal promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression.

[0053] There are several types of regulatory regions, including those that are developmentally regulated, inducible or constitutive. A regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory regions that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within the plant as well. Examples of tissue-specific regulatory regions, for example see-specific a regulatory region, include the napin promoter, and the cruciferin promoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (see US 7,125,978, which is incorporated herein by reference). [0054] An inducible regulatory region is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible regulatory region to activate transcription may be present in an inactive form, which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory region may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible regulatory elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, LR.P., 1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference). Examples, of potential inducible promoters include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48,89-108; which is incorporated by reference), steroid inducible promoter (Aoyama. T. and Chua, N.H.,1997, Plant 1. 2, 397-404; which is incorporated by reference) and ethanol-inducible promoter (Salter, M.G., et al, 1998, Plant lOurnal 16, 127-132; Caddick, M.X., et al,1998, Nature Biotech. 16, 177-180, which are incorporated by reference) cytokinin inducible IB6 and CKI 1 genes (Brandstatter, I. and K.ieber, 1.1.,1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274,982-985; which are incorporated by reference) and the auxin inducible element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is incorporated by reference).

[0055] A constitutive regulatory region directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript (Odell et al., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991, Plant Cell, 3: 1155-1165), actin 2 (An et al, 1996, Plant J., 10:

107-121), or tms 2 (U.S. 5,428,147, which is incorporated herein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et ai, 1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637- 646), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995, Plant Mol. Biol. 29: 995-1004). [0056] The term "constitutive" as used herein does not necessarily indicate that a gene under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is often observed. Constitutive regulatory elements may be coupled with other sequences to further enhance the transcription and/or translation of the nucleotide sequence to which they are operatively linked. For example, the

CPMV-HT system is derived from the untranslated regions of the Cowpea mosaic virus (CPMV) and demonstrates enhanced translation of the associated coding sequence. By "native" it is meant that the nucleic acid or amino acid sequence is naturally occurring, or "wild type". By "operatively linked" it is meant that the particular sequences, for example a regulatory element and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences. [0057] "Expression cassette" refers to a nucleotide sequence comprising a nucleic acid of interest under the control of, and operably (or operatively) linked to, an appropriate promoter or other regulatory elements for transcription of the nucleic acid of interest in a host cell.

[0058] The one or more chimeric genetic constructs of the present invention can further comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA precursor.

Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. Non-limiting examples of suitable 3' regions are the 3' transcribed nontranslated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, plant genes such as the soybean storage protein genes, the small subunit of the ribulose-I, 5-bisphosphate carboxylase gene

(ssRUBISCO; US 4,962,028; which is incorporated herein by reference), the promoter used in regulating plastocyanin expression, described in US 7, 125,978 (which is incorporated herein by reference).

[0059] One or more of the chimeric genetic constructs of the present invention may also include further enhancers, either translation or transcription enhancers, as may be required. Enhancers may be located 5' or 3' to the sequence being transcribed.

Enhancer regions are well known to persons skilled in the art, and may include an ATG initiation codon, adjacent sequences or the like. The initiation codon, if present, may be in phase with the reading frame ("in frame") of the coding sequence to provide for correct translation of the transcribed sequence.

[0060] The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see for example Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIE, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct DNA uptake, the use of liposomes, electroporation, for example using protoplasts, micro-injection, microprojectiles or whiskers, and vacuum infiltration. See, for example, Bilang, et al. (Gene 100: 247-250 (1991), Scheid et al. (Mol. Gen. Genet. 228: 104- 112, 1991), Guerche et al. (Plant Science 52: 111-116, 1987), Neuhause et al. (Theor. Appl Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987); Howell et al. (Science 208: 1265, 1980), Horsch et al. (Science 227: 1229-1231, 1985), DeBlock et al, Plant Physiology 91: 694-701, 1989), Methods for Plant Molecular Biology (Weissbach and Weissbach, eds., Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), Liu and Lomonossoff (I Virol Meth, 105:343-348, 2002,), U.S. Pat. Nos. 4,945,050; 5,036,006; and 5,100,792, U.S. patent application Ser. Nos. 08/438,666, filed May 10, 1995, and 07/951,715, filed Sep. 25, 1992, (all of which are hereby incorporated by reference).

[0061] Transient expression methods may be used to express the constructs of the present invention (see Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348; which is incorporated herein by reference). Alternatively, a vacuum- based transient expression method, as described by Kapila et al., 1997, which is incorporated herein by reference) may be used. These methods may include, for example, but are not limited to, a method of Agro-inoculation or Agro-infiltration, syringe infiltration, however, other transient methods may also be used as noted above. With Agro-inoculation, Agro-infiltration, or synringe infiltration, a mixture of Agrobacteria comprising the desired nucleic acid enter the intercellular spaces of a tissue, for example the leaves, aerial portion of the plant (including stem, leaves and flower), other portion of the plant (stem, root, flower), or the whole plant. After crossing the epidermis the Agrobacteria infect and transfer t-DNA copies into the cells. The t-DNA is episomally transcribed and the mRNA translated, leading to the production of the protein of interest in infected cells, however, the passage of t-DNA inside the nucleus is transient.

[0062] The peptide or protein target, the affinity trapping agent, or both the peptide or protein target and the affinity trapping agent, may be expressed stably, or they may be transiently co-expressed. Furthermore, one of the peptide or protein target, or the affinity trapping agent may expressed stably, while the other of the peptide or protein target, or the affinity trapping agent may be expressed transiently.

[0063] To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers are known to one of skill in the art and may include enzymes that provide for resistance to chemicals such as an antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as phosphinothrycin, glyphosate, chlorosulfuron, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (beta- glucuronidase), or luminescence, such as luciferase or GFP, may be used. [0064] Also considered part of this invention are transgenic plants, plant cells or seeds containing the chimeric gene construct of the present invention. Methods of regenerating whole plants from plant cells are also known in the art. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques. Transgenic plants can also be generated without using tissue cultures.

Intracellular scFvsfor targeted expression in plant cells

[0065] Due to the ease for localizing a fluorescent protein in the plant cell, green fluorescent protein (GFP) was used as an non-limiting example of a protein of interest, or as an epitope tag fused to a plant- made pharmaceutical (PMP). To further exemplify the present invention, scFv specific for GFP were used. However, it is to be understood that any peptide or protein having an affinity binding capacity to a specific target may be used as described herein.

[0066] Two methods were used for isolating functional scFv specific for GFP. In the first approach, the scFv was generated from the variable regions of a high affinity monoclonal antibody specific for GFP. The V_H and V_L regions were amplified by reverse transcription (RT)-PCR of mRNA from an original hybndoma and cloned as a scFv. In the second approach, phage display technology was employed to isolate two GFP-binding scFvs using the spleen of a rabbit immunized with purified GFP as starting material. scFvs generated from a GFP-specific mAbs were screened and scFvs were selected for further use based on increased specificity for GFP and GFP spectral variants, and the absence of proteolytic cleavage when produced in a plant expression system. A FlagHis tag and appropriate targeting signals were added at the N- and C-terminal ends of the scFv to produce the constructs illustrated in Figure 1. The scFv constructs were stably or transiently expressed in different plants, either as a soluble protein, or as a membrane protein, for example but not limited to a type II membrane protein comprising a signal-anchor sequence targeted to the ER lumen with its C-terminal domain. Studies to analyze the capacity of this scFv for intracellular targeting of a protein of interest were performed using transient co-expression of the target protein, an inhibitor of silencing HcPro, and the appropriate scFv, using Agrobacterium vacuum infiltration of intact N. benthamiana leaves as described in Vezina et ah, (2009; which is incorporated herein by reference).

[0067] Described herein are soluble or membrane bound scFv localized respectively in the ER or in the Golgi apparatus to target either soluble or membrane bound proteins inside these specific subcellular compartments.

Production and selection of a scFv specific for GFP.

[0068] Using a phage display antibody library, a panel of anti-green fluorescent protein (GFP) scFvs were generated. Two of these scFv's (scFvl and scFv2) were selected for further studies. The spleen of a New Zealand rabbit immunized with GFP was used for mRNA extraction and generation of a phage displayed, scFv antibody, library. Purified GFP was used for panning of this scFv library and selection of scFvl and scFv2, in accordance with techniques commonly used in the art. A third anti-GFP scFv (scFv3,) was cloned from a monoclonal antibody isolated from a murine hybridoma. The tobacco chitinase signal peptide (SP) was fused at the N-terminus of each scFv sequence to direct these antibody fragments into the secretory pathway. Retention of the scFv to the ER was achieved by adding an HDEL peptide (Gomord et ah, 2004. Conley et /,.2010) at the C-terminus. A FLAG tag and a (His)6 sequence were spliced between the scFv sequence and the HDEL tetrapeptide to facilitate immunodetection and purification. These constructs are shown in Figure 1 (soluble ER-scFv). All three scFvs were transiently expressed in agroinfiltrated Nicotiana benthamiana leaf epidermal cells.

[0069] Even though retaining recombinant proteins in the ER increases protein stability due to the low abundance of proteolytic enzymes in this compartment, some proteolytic processing events have been reported. For example, Badri et al. (2009) describes sequential removal of specific amino acids at the N and C termini of bovine plasma protein aprotinin expressed in leaves of transgenic potato lines by endogenous peptidases proteins and that accumulated into the ER. As shown in Figure 2, unintended protein processing of SolER-scFv (scFvl and scFv2) was also observed. Truncated forms of scFvl and scFv2 were observed on a western blot using crude protein extracts prepared from N. benthamiana leaves collected six days after agroinfiltration. However, SolER-scFv form, scFv3, was not truncated. Without wishing to be bound by theory, proteolytic cleavage of scFvl and scFv2 may explain why the affinity for GFP was reduced when compared to GFP affinity exhibited by scFv3 (data not shown).

[0070] As illustrated Figure 3, scFv3 cross reacts with GFP and its spectral variants, the cyan fluorescent protein (CFP), and the yellow fluorescent protein (YFP), but not with monomeric red fluorescent protein (mRFP; this provides an internal control for binding specificity as illustrated below). In contrast, scFvl and scFv2 cross react with GFP, CFP, YFP and mRFP (data not shown). For these reasons, scFv3 was selected for further studies.

ScFv- mediated in vivo affinity trapping allows accumulation of soluble and membrane proteins in the different compartments of the secretory pathway.

[0071] The scFv -mediated in vivo affinity trapping strategy described here is an attractive alternative to the generation of chimeric forms of the protein of interest fused with different targeting signals and can be used to determine a suitable subcellular location for quality, activity, yield or a combination thereof.

[0072] Expression of a soluble or membrane -bound scFv allows efficient ER retention or controlled sub-Golgi accumulation of a soluble or membrane-bound antigen. For example, affinity trapping in the ER has been observed of either soluble or membrane bound GFP using a soluble scFv targeted to the ER (see Figure 3B, 4 D, 4J, 5B, 5D, 5F, 5H, 6D, 6G, 7D, 7 J, 8D, 8G). Membrane targeting signals (Pagny et ah, 2003; Saint-Jore et ah, 2002; Saint-Jore-Dupas et ah, 2006; Boulaflous et ah, 2009) fused to scFv have been used to produce affinity trapping agents that target different sub-Golgi compartments. The scFv fusions can be used to move or retain a membrane bound target protein from one Golgi domain to another. For example, a trans Golgi protein has been targeted to the cis Golgi stack when co-expressed with a scFv located in this compartment. The affinity trapping strategy descried herein allows for the accumulation of a soluble protein in specific Golgi cisternae.

[0073] In the absence of available signal sequences to target soluble proteins into the lumen of specific Golgi cisternae, the affinity trapping methods described herein, for example, scFv-mediated in vivo targeting, provides an approach at accumulating soluble proteins into the Golgi.

[0074] . As a non-limiting example, affinity trapping using soluble or membrane bound versions of a GFP- specific scFv may be used to predict the intracellular transport or the topology of membrane or soluble proteins in the secretory pathway.

A soluble ER-scFv retains a secreted protein into the ER.

[0075] When a secreted form of enhanced CFP (eCFP) fused with the signal peptide of tobacco chitinase (SP-eCFP), (Figure 1) is transiently expressed in N. benthamiana leaf epidermal cells; it is secreted and accumulates in the apoplast (Figure 4A, 4G). SP-eCFP subcellular localization is distinct from that of mRFP fusions used either as ER- (Figure 4B, 4C) or as trans Golgi markers (Figure 4H).

[0076] When SP-eCFP was transiently co-expressed with SolER-scFv, SP-eCFP accumulates in the ER (Figure 4D) and co-localizes with the ER marker mRFP-HDEL (Figure 4F). As shown in Figure4 J-L, this scFv-mediated in vivo ER targeting is clearly distinct from the trans-Golgi network.

[0077] The subcellular localization of the mRFP tagged trans-Golgi marker is not affected by the co-expression with the GFP-specific SolER-scFv (Figure 4K), which confirms that the scFv3 used in this study reacts with GFP and eCFP but not with mRFP. Preservation of the Golgi apparatus (Figure 4L) structure illustrates that ER retention observed for SP-eCFP co-expressed with SolER-scFv is due to specific affinity trapping and not to a general blockage of secretion due to the accumulation of the scFv in the ER lumen. As summarized Figure 11 A, these data demonstrate that a soluble scFv retained in the ER promotes ER retention of it soluble antigen counterpart. This strategy allows accumulation of a soluble secreted protein in the ER lumen without the need of a fusion of a K/HDEL, ER retrieval signal at its C-terminal end.

A soluble ER-scFv relocalizes Golgi membrane proteins into the ER.

[0078] Three GFP fusions proteins were used as either a cis Golgi marker (Figure 1 and 5A), a medial Golgi marker (Figure 1 and 5C), or a trans Golgi marker (Figure 1 and 5G). In these fusions, the sub-Golgi localization signals fused to GFP were derived respectively from, Glycine max mannosidase I (Man99; Saint-Jore-Dupas et al, 2006), Arabidopsis thaliana β1,2 xylosyltransferase (XYLT35; Pagny et al., 2003) and rat sialyltransferase (ST52; Saint-Jore et al., 2002). [0079] When these reporter membrane proteins were transiently expressed simultaneously with the soluble GFP-specific scFv retained in the ER lumen (SolER- scFv), they accumulated in the ER membrane, as illustrated from the ER pattern observed in these conditions (Figure 5B, D, H), in contrast with the typical Golgi pattern shown when these Golgi markers are expressed alone (Figure 5A, C, G). This scFv-mediated relocalization of Golgi membrane proteins is scFv- specific. The specificity of this relocalization is illustrated figure 5F when out of two medial Golgi markers made of either GFP (Figure 5C) or mRFP (Figure 5E) fused to the same signal for specific anchoring of the marker into the membranes of the medial Golgi, only the GFP fusion Golgi marker is relocalized into the ER when co-expressed with the GFP-specific SolER-scFv (Figure 5D) while the mRFP is not (Figure 5F).

[0080] Therefore, a soluble scFv located in the ER has the capacity to retain in the ER membrane proteins located either in the cis, medial or trans Golgi cisternae

(Figure 1 IB). Non limiting examples for the application of this strategy include i) preventing enzymatic modification occurring in the Golgi by a scFv-mediated depletion of the modifying enzyme from the Golgi network; ii) enhancing the ER with part of the Golgi-specific enzymatic machinery; and iii) targeting and storing a membrane-bound biopharmaceutical in the ER.

A membrane-bound Golgi-scFv mediates accumulation of soluble or membrane- bound proteins into the Golgi. [0081] The first 99 amino acids of G. max mannosidase I (Man99), were previously shown to be sufficient for targeting a reporter protein in the cis Golgi membrane (Saint-Jore-Dupas et al., 2006). Man99 was fused at the N-terminal end of a GFP- specific scFv to produce a membrane-bound Golgi scFv (membrane Golgi scFv; mb Golgi scFv) as illustrated Figure 1. The capacity of the membrane-bound scFv to relocalize soluble or membrane-bound target proteins, was examined.

[0082] As illustrated Figure 6, the soluble protein SP-eCFP, that accumulates in the apoplast when expressed alone in tobacco leaf cells (Figure6A) is relocalized in the Golgi apparatus when co-expressed with mb Golgi scFv (Figure 6D, 6G) where it colocalizes with a trans Golgi marker (Figure 61). The enlarged portion of the merged picture presented Figure 61 illustrates that SP-ECFP and a trans Golgi marker accumulate respectively in early and late domains in each Golgi stack. This approach demonstrates accumulation of a soluble protein in a sub-Golgi domain.

[0083] To test the ability of a membrane-bound Golgi scFv to relocate a membrane protein using scFv-mediated targeting, a fusion membrane protein (R/LGCS90-GFP), (described in Boulaflous et al, 2010; which is incorporated herein by reference) previously shown to co-localize with a trans Golgi marker (Boulaflous et al, 2009, which is incorporated herein by reference) was used (Figure 7 panels G, J). As illustrated in Figure 7, panels J-L, it is possible to target PJLGCS90-GFP into the early Golgi by co-expressing it with a scFv anchored in the cis Golgi compartment.

[0084] Together, these results illustrate that a membrane-bound Golgi scFv can retain a secreted soluble protein or relocalize a membrane protein into a specific Golgi subcompartment (see Fig 11C for schematic illustration). The present affinity trapping method provides for the identification of suitable sub Golgi localization, for example, for heterologous glycosyltransferases or glycosidases to generate PMP glycoforms of high therapeutic efficiency. Furthermore, this affinity trapping method can also allow for the accumulation of a biopharmaceutical as the target protein into a defined sub-Golgi compartment for example, thus influencing, such as by limiting or facilitating, Golgi- specific maturations of the biopharmaceutical.

A membrane-bound ER-scFv retains Golgi membrane proteins into the ER [0085] As previously shown in Saint-Jore-Dupas et al. ( 2006 ), an eCFP fusion targeted to the early Golgi membranes and an mRFP fusion targeted to the medial Golgi membranes may be used as Golgi markers since they co-localize in the Golgi but only partially overlap in each dictyosomes, (Figure 7, panel C). When a membrane- bound ER scFv is co expressed with these two Golgi proteins, the membrane- bound version of eCFP does not accumulate anymore in the Golgi but is retained in the ER membranes. In contrast the localization of the mRFP Golgi marker remains unchanged in the same conditions (Figure 7, panels D-F) Therefore, a membrane- bound scFv located in the ER membranes has the capacity to retain Golgi resident proteins in the ER membranes.

[0086] Non limiting applications of this strategy include i) preventing an enzymatic modification occurring in the Golgi by a scFv-mediated depletion of the modifying enzyme from the Golgi network; ii) enhancing the ER with part of the Golgi- specific enzymatic machinery; and iii) targeting and store a membrane-bound

biopharmaceutical in the ER.

ScFv-mediated targeting of a pharmaceutical protein

[0087] In this application of affinity trapping, the biopharmaceutical of interest is Derp2, one of the major allergens of the house dust mite Dermatophagoides pteronyssinus. This allergen was previously produced in tobacco plants and in suspension-cultured BY2 tobacco cells (Lienard et al., 2006). For the current application, a N-glycosylation site was generated in the allergen sequence (Derp2Y) to study both the scFv-mediated relocalization of this biopharmaceutical protein and the effects of this approach on its post-translational maturations. Based on previous structural studies on Derp2 (Mueller et al, 1997, 1998), one asparagine residue (Asn 120) was identified as a good candidate for Ν-glycosylation due to its high accessibility at the surface of the folded allergen. To generate such a potential glycosylation site in the Derp2 sequence, we thus engineered a Vall22Thr mutation to allow N-glycan attachment to Asn 120.

[0088] As illustrated in Figure 8 when transiently expressed in tobacco leaves, Derp2Y is secreted and appears as a diffuse fluorescence at the surface of the cells (Figure 8A). In contrast, when Derp2Y is transiently co-expressed with a specific ER scFv, the recombinant allergen is retained and accumulates in the ER lumen (Figure 8D) where it perfectly co localizes with the ER marker mRFP-HDEL (Figure 8F).

[0089] It is also possible to accumulate the recombinant allergen into the Golgi, such as, in the present case into the early Golgi, by co-expressing Derp2Y with a specific scFv anchored in the cis Golgi membranes for example, where it only partially co- localizes with a trans Golgi marker (see Figure 81).

[0090] Another application to the ER retention of PMPs is to prevent posttranslational modifications occurring even more downstream in the secretory pathway, including for example proteolytic cleavages and complex N-glycan biosynthesis. Interestingly, and in contrast with Derp2, Derp2Y reacts with antibody probes specific for complex plant N-glycans and with concanavalin A (ConA), a lectin specific for high mannose type N-glycans (Figure 9A). These results indicate that different Derp2Y glycoforms are secreted by tobacco cells. Some of these glycoforms have high mannose type N- glycans reacting with ConA, while the others have endo-H resistant complex type N- glycans containing at least one of the tree immunogenic glycoepitopes typical for plant glycoproteins: the al, 2- xylose-, i,3-fucose- containing glycoepitopes and the Le^a epitope. In contrast, when Derp2Y is co-expressed with a specific ER scFV, the glycosylated allergen remains in the ER (Figure 8D) and its N-glycan remains under a high mannose form cleaved by endoH (Figure 9B).

[0091] Therefore, the co-expression of a pharmaceutical protein fused with a tag and a tag-specific scFv allows, for example, for investigating suitable subcellular localization for yield and quality of the pharmaceutical protein. This approach is not limited to transient expression in N. Benthamiana or N. tabacum, as illustrated Figure 10 where stable (B, C) or transient (A, D) co-expression of Derp2Y with transient expression of a specific ER-scFv lead to the affinity trapping of Derp2 in different plant expression systems such as Medicago sativa ( Fig 10A), suspension-cultured BY2 tobacco cells (Fig 10B and C) and Arabidopsis thaliana ( Fig 10D).

[0092] Compared to the usual approach used for targeted expression where pharmaceutical proteins or maturation enzymes studied are fused to different targeting signals, the present method provides for a same tag-specific scFv modified for targeting in different compartments of the secretory pathway, which can be used for example to investigate activity, quality and yield for any protein of interest harbouring a same tag.

ScFv -mediated in vivo targeting: many applications in molecular farming

[0093] The examples provided herein illustrate many applications of using an affinity trapping agent, for example scFv-mediated targeting, in molecular farming. With GFP, and using a specific scFv, it is possible to investigate which cellular compartment is the most appropriate for qualitative and quantitative optimization of a PMP production. Targeting and purification tags are removed from a PMP to reduce any immunogenicity and related concerns raised by drug regulatory agencies. As exemplified here using GFP as a peptide or protein of interest, and an scFv against this specific target, there is no need for a targeting or purification tag on the peptide or protein of interest.

[0094] The use of a tag and a tag-specific scFv, makes scFv-mediated targeting a general tool for optimization of PMP production. For example it is shown here that a secreted and glycosylated form of the recombinant allergen Derp2 fused to a tag, (GFP being used here as a tag) can be immunotrapped in the ER when co-expressed with an ER- tag-specific scFv. An advantage of using GFP as an epitope tag in the present study is that the efficiency of the procedure for relocalization of the protein of interest can be easily illustrated using confocal microscopy. For alternate applications of the immunotrap technology in molecular farming, smaller peptide epitope tags like the FLAG- tag (Einhauer and Jungbauer 2001) which can be used in conjunction with an anti-FLAG scFv cloned from one of several anti-FLAG monoclonal antibodies available (Brizzard et ah, 1994), may be used.

[0095] Previous results obtained using recombinant glycoproteins fused with a H/KDEL tag were efficiently retained in the ER (Sriraman et ah, 2004, Petrucelli et ah, 2006), after scFv- mediated ER retention Derp2Y carries only high-mannose N- glycans whereas, Derp2Y carries complex N-glycans when secreted in the intercellular space. In addition to the positive impact of ER retention on the yield of Derp2Y, different Derp2Y glycoforms may also be generated using scFv-mediated targeting through co expression of different N-glycan maturation enzymes fused with GFP. This means that using a same family of tag-specific scFv different combination of PMP and PMP maturation enzymes could be investigated simultaneously in a same subcellular compartment without the need of extensive generation of fusions for each partner. Interestingly, this approach not only saves time, it is also safer, indeed the efficiency of targeting does not need to be validated for each fusion protein but only once for the different sub cellular forms of the tag-specific scFv.

[0096] The present invention will be further illustrated in the following examples. Example 1: Materials

[0097] Golgi and ER fluorescent marker used in this study were described in Saint- Jore-Dupas et al., 2006 (Cis Golgi marker :MAN99-GFP ; Medial Golgi marker : XylT35-GFP ; and Trans Golgi marker : ST52-GFP and ST52-mRFP); and

Boulaflous et al, 2009 (R/LGCS90-GFP,XylT35-mRFP ). The Der p 2Y

glycoallergens was generated from Der p 2, the major allergen from

Dermatophagoides pteronissinus as described in Sourrouille et al., (submitted). The SP-CFP was a generous gift of Pr Dolors LUDEVID group at CSIC Barcelona.

Example 2. Construction of plant expression cassette containing the anti-GFP scFv [0098] The heavy- and light-chain cDNAs of a monoclonal antibody directed against

GFP (provided by Prof. P.Billiald, Museum Histoire Naturelle, Paris, France) were used for generation of anti-GFP scFv-cDNA. The VH and VL fragments were amplified independently by polymerase chain reaction (PCR) using domain specific primers described in Table 1. For each domain, one primer contained an overlapping sequence to form the VH and VL connecting linker and a BamHl endonuclease site was used in conjunction with a primer containing both a Kpnl site and a chitinase signal sequence encoding sequence or a Spel restriction site and a FlagHisHDEL Tag encoding sequence. For expression in plant cells, the chitinase signal peptide encoding sequence, was added at the 5' end of the variable domain of the heavy chain using the primers ForVH-Kpnl and RevVh-Linker-BamHI (Table 1) and pGscFV as a template and then cloned into the Kpnl and BamH 1 endonuclease sites of pBLTI121 (Pagny et al., 2003) to give the pBLTIVh vector. Then, a sequence encoding the KDEL C- terminal and the FLAG-His Tag was added by PCR for retention of the ScFv in the ER, using the primers ForVl-linker-Bam Hi and RevVl-TagHDEL-Spel and the pGscFV as a template. This product was cloned in the pBLTIVh vector at the BamHl and Spel endonuclease sites to give the pBLTISolErScFv vector.

[0099] Membrane Golgi-ScFv was amplified using primers ForMan99-BamHl and RevTag-XXX and the pBLTIsolER scFv vector as a template. The amplification product was subcloned in the PBLTI121.

Table 1: Oligonucleotide primers used in this study

Example 3. Agrobacterium -mediated transient expression

[00100] A. tumefaciens was cultured at 28°C until the stationary phase

(approximately 20h), washed and resuspended in infiltration medium (MES 50mM pH5.6, glucose 0.5% (w/v), Na₃P0₄ 2 mM, acetosyringone 100 μιη from 10 mM stock in absolute ethanol) (Saint-Jore et al, 2002). The bacterial suspension was pressure injected into the abaxial epidermis of N. benthamiana, G. max or L. esculentum leaves using a 1-mL plastic syringe by pressing the nozzle against the lower leaf epidermis as previously described in Neuhaus and Boevink, (2001). The plants were incubated for 3 days at 20-25°C unless specified in the figure legend. For transient expression in tomato and soybean, the same syringe infiltration procedure was followed but plants were analysed 2 days after infiltration. [00101] Stable transformation of BY-2 tobacco (Nicotania tabacum) cells was performed as described previously (Gomord et al., 1998).

Example 4. Protein extraction, SDS-PAGE and western analysis

[00102] Crude protein extracts were obtained from agroinfiltrated N.

benthamiana leaves ground with two volumes of prewarmed denaturation buffer (Tris 60mM, pH6.8, SDS, 1%, glycerol, 10%, β-mercaptoefhanol 2%), boiled for 5 minutes, and centrifuged 15 minutes at 8000g. The supernatant was directly analyzed by SDS- PAGE in 15% polyacrylamide gels. After SDS-PAGE, proteins were transferred from the gel onto a nitrocellulose membrane and immunodetected using a polyclonal rabbit immunserum directed against HisFlag tag (Pagny et al., 2000) at a 1 : 1000 dilution followed by incubation with goat anti-rabbit IgG antibodies coupled to horseradish peroxidase (Bio-Rad, Marnes la Coquette, France) diluted 1 :3000 in 1% gelatin in TBS for lh at room temperature. After incubation with HRP-conjugated anti-rabbit secondary antibody, Immunoreactive signals were detected with 4-chloro-l-naphthol.

Example 5. Ν- Glycan structural analysis [00103] After transfer from SDS-PAGE gels onto nitrocellulose membranes, glycoproteins were analyzed by either lectin affinoblotting (affinodetection) using the Con A/peroxidase system specific for high-mannose-type N-linked glycans (Faye and Chrispeels, 1985), or immunoblotting with antibodies specific for l,2-xylose-, β1,3- fucose- or Lewis a-containing glycoepitopes on plant complex N-glycans (Faye et al., 1993, Fitchette et al. 1997). Briefly, for affinodetection after protein transfer, the membranes were saturated for lh in TBS buffer (20 mM Tris-HCl, pH 7.4, containing 0.5M NaCl) containing 0.1% Tween 20 (TTBS), followed by two successive incubations at room temperature in 25μg.mL^"1 concanavalin A (Sigma- Aldrich, St Quentin Fallavier, France) in TTBS containing ImM MgCl₂ and ImM CaCl₂ for 1.5h, and then in 50 μ g.mL^"1 horseradish peroxidase (Sigma-Aldrich) in TTBS for lh. After several washes in TTBS and a short rinsing in TBS , glycoproteins are detected on the membrane using 4-chloro-l-naphtol as substrate for horseradish peroxidase.

Example 6. Deglycosylation with endoglycosidase H

[00104] For deglycosylation with Endo H, proteins were denatured by incubating at 100°C for 5 min in the presence of 1% SDS (w/v). After a fivefold dilution with 150 m sodium acetate, pH 5.7, the protein sample was incubated for 6h at 37°C with 10 mU Endo H. After the deglycosylation reaction an equal volume of twice concentrated electrophoresis sample buffer was addded before analysis using SDS-PAGE.

Example 7. Confocal Laser Scanning Microscopy

[00105] Cells expressing GFP/mRFP fusions were imaged using a Leica TCS

SP2 AOBS confocal laser scanning microscope (CLSM). Single color imaging of GFP was realized using a 488-nm argon ion laser line and the fluorescence was recorded by a photomultiplier set up for 493-538 nm. Dual-color imaging of cells co-expressing GFP and mRFP was performed using simultaneously a 488-nm argon ion laser line and a HeNe 543 nm laser line. Fluorescence signals were separated using the acousto- optical beam splitter (AOBS) and GFP emission was detected in photomultiplier 2 (493-538 nm) whereas mRFP was collected in photomultiplier 3 (580-620 nm). The power of each laser line, the gain and the offset were identical for each experiment so that the images were comparable. Appropriate controls were performed to exclude the possibility of cross talk between the two fluorochromes before the image acquisition.

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[00106] All citations are hereby incorporated by reference.

[00107] The present invention has been described with regard to one or more embodiments. However, the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid encoding an affinity trapping agent, the affinity trapping agent comprising a peptide or protein ligand having a binding affinity for a protein of interest, fused to a heterologous subcellular compartment targeting signal.

2. The nucleic acid of claim 1, wherein the peptide or protein ligand is selected from the group of single chain variable fragment (scFv), an scFv variant, bivalent scFv, trivalent scFv, Protein A, Protein L, Protein G, Protein A/G.

3. The nucleic acid of claim 1 or 2, wherein the heterologous subcellular compartment targeting signal targets the affinity trapping agent to a subcellular compartment selected from the group of endoplasmic reticulum, Golgi network, chloroplast, cytosol, mitochondria, nucleus and peroxisome.

4. The nucleic acid of claim 3, wherein the Golgi network further comprises cis- Golgi, median-Golgi, and trans-Golgi.

5. The nucleic acid of any one of claims 1 to 4, wherein the affinity trapping agent further includes a FLAG tag epitope, poly (His), Strep II, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag.

6. A method for targeting the immobilization of a recombinant peptide or protein of interest to a subcellular compartment of a cell comprising,

i) introducing a nucleotide sequence encoding the recombinant peptide or protein of interest into the cell;

ii) introducing the nucleic acid encoding the affinity trapping agent of any one of claims 1 to 5 into the cell; and

iii) co-expressing the nucleotide sequence encoding the recombinant peptide or protein of interest and the nucleic acid encoding the affinity trapping agent in the cell to form a complex and targeting the immobilization of the recombinant peptide or protein of interest.

7. The method of claim 6, wherein the recombinant peptide or protein of interest is either soluble or membrane bound.

8. The method of claim 6 or 7, wherein the recombinant peptide or protein of interest is a plant made pharmaceutical, an enzyme, or a protein maturation enzyme.

9. The method of any one of claims 6 to 8, wherein the recombinant peptide of interest comprises a FLAG tag epitope, poly (His), Strep Π, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag.

10. The method of any one of claims 6 to 9, wherein

i) the recombinant peptide or protein of interest is soluble, and the affinity trapping agent is membrane bound;

ii) the recombinant peptide or protein of interest is membrane bound, and the affinity trapping agent is membrane bound

iii) the recombinant peptide or protein of interest is membrane bound, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal; or

iv) the recombinant peptide or protein of interest is soluble, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal.

11. The method of any one of claims 6 to 10, wherein the affinity trapping agent comprises a FLAG tag epitope a FLAG tag epitope, poly (His), Strep Π, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag, and the epitope or tag is used to purify the complex.

12. The method of any one of claims 6 to 11, wherein: a) in the step of introducing, step i), the nucleotide sequence is introduced into the cell in a transient manner;

b) in the step of introducing, step i), the nucleic acid sequence is introduced into the cell in a transient manner; or

c) in the steps of introducing, step i) and step ii), the nucleotide sequence and the nucleic acid sequence are introduced into the cell in a transient manner.

13. The method of any one of claims 6 to 12, wherein the cell is a plant cell.

14. The method of claim 13, wherein the plant cell is a Nicotian benthamiana cell, a Nicotiana tabaccum cell, a Medicago sativa cell, or an Arabidopsis thaliana cell.

15. A method for targeting the immobilization of an endogenous peptide or protein of interest to a subcellular compartment of a cell comprising,

i) introducing the nucleic acid encoding the affinity trapping agent of any one of claims 1 to 5 into the cell, the affinity trapping agent having a binding affinity for the endogenous peptide or protein of interest; and

ii) expressing the nucleic acid encoding the affinity trapping agent in the cell to form a complex and targeting the immobilization of the endogenous peptide or protein of interest.

16. The method of claim 15, wherein the endogenous peptide or protein of interest is either soluble or membrane bound.

17. The method of claim 15 or 16, wherein the endogenous peptide or protein of interest is an enzyme endogenously expressed by the cell.

18. The method of any one of claims 15 to 17, wherein

i) the endogenous peptide or protein of interest is soluble, and the affinity trapping agent is membrane bound;

ii) the endogenous peptide or protein of interest is membrane bound, and the affinity trapping agent is membrane bound

iii) the endogenous peptide or protein of interest is membrane bound, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal; or

iv) the endogenous peptide or protein of interest is soluble, and the affinity trapping agent is soluble and comprises a subcellular compartment targeting signal.

19. The method of any one of claims 15 to 18, wherein the affinity trapping agent comprises a FLAG tagepitope, poly (His), Strep Π, chitnase binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), covalent yet dissociable NorpD peptide (CYD), heavy chain Protein C (HPC), V5-tag, c-myc-tag, hemagglutinin (HA)-tag, and the epitope or tag is used to purify the complex.

20. The method of any one of claims 15 to 19, wherein:

a) in the step of introducing, step i), the nucleic acid sequence is introduced into the cell in a transient manner.

21. The method of any one of claims 15 to 20, wherein the cell is a plant cell.

22. The method of claim 21, wherein the plant cell is a Nicotian benthamiana cell, a Nicotiana tabaccum cell, a Medicago sativa cell, or an Arabidopsis thaliana cell.