WO2019063992A1

WO2019063992A1 - Protease inhibitors

Info

Publication number: WO2019063992A1
Application number: PCT/GB2018/052731
Authority: WO
Inventors: Friederike GROSSE-HOLZ; Renier VAN DER HOORN
Original assignee: Oxford University Innovation Limited
Priority date: 2017-09-26
Filing date: 2018-09-26
Publication date: 2019-04-04
Also published as: GB201715589D0

Abstract

Proteases limit recombinant protein production in molecular pharming since endogenous plant proteases degrade foreign protein through partial hydrolysis. Protease inhibitors are used for production of recombinant proteins in plants. Genetically modified plants expressing the protease inhibitors into their apoplast provide for high level of recombinant protein accumulation which can be isolated from the plant material, usually leaves. Three protease inhibitors are described: HsTIMP2 which is a human metalloprotease inhibitor; NbPR4 which is a homolog of CaPR4 which was recently shown to be a protease inhibitor; and NbPotI which was not known previously to be a protease inhibitor, nor was it linked to transient protein expression. These protease inhibitors are used in plants as tools for increasing desired recombinant protein expression levels. Expression constructs encoding the protease inhibitors and the recombinant proteins are described..

Description

PROTEASE INHIBITORS

FIELD OF THE INVENTION

The invention relates to protease inhibitors for the production of recombinant proteins in plants, methods of expressing protein in plants using the protease inhibitors of the invention and modified plants capable of a high level of recombinant protein accumulation.

BACKGROUND TO THE INVENTION

Plants are an attractive system for heterologous protein expression, because of their potential for scalability, low production and maintenance costs, reduced requirements for investment in infrastructure, potential for using lower value land in production and in the case of therapeutic proteins, fewer problems associated with post-translational modifications, processing and contamination of the protein fraction that are often associated with prokaryotic expression systems. However, the potential of plants as expression systems for proteins of interest has not yet been fully realised, primarily a consequence of lower level of recombinant protein accumulation achieved in plant systems.

Apart from their conventional purpose of food production, plants have been studied in modern biotechnology to produce antibodies and vaccines. The production of pharmaceutical vaccines and antibodies using plants is known as "molecular pharming". In agricultural production, crops are lost through diseases, and molecular pharming is limited by recombinant protein yields. Proteases play an important role in both plant immunity, combating diseases, and in molecular pharming, limiting recombinant protein production through proteolytic degradation. Understanding proteases can therefore have applications in boosting both plant immunity and recombinant protein yields.

In the last 20 years, plants have become an attractive tool for recombinant protein production. The advantages of plant based recombinant proteins include cheap, robust production pipelines and low risk of contamination with human pathogenic bacteria or viruses. Also, plants can perform post-translational modifications such as glycosylation and amidiation, which do not occur in prokaryotic systems commonly used for vaccine and antibody production. Transient expression in plants facilitates the production of vast amounts of recombinant protein in a shorter period compared to mammalian systems. In this system, genes of interest are expressed using Agrobacterium tumefaciens, which is a soil bacterial pathogen. Engineered A. tumefaciens can deliver foreign DNA sequences into the plant cell. Nicotiana benthamiana has been used as a model system for recombinant protein production in molecular pharming since it has been extensively studied as a model plant organism. Transient expression in Nicotiana benthamiana is a quickly developing new protein expression platform to quickly and safely produce humanized glycoproteins in plants. Examples: Medicago (Canada) can produce 10 million influenza vaccins in 6 weeks; Leaf Expression Systems (Norwich) is BBSRC-supported facility that will use this platform to produce proteins for pharma trials; iBio and KBP (USA) have produced ZMAPP antibodies during the Ebola crisis.

Proteases limit recombinant protein production in molecular pharming since endogenous plant proteases degrade foreign protein through partial hydrolysis. Previous studies have shown that the plant proteolytic machinery can degrade full-size antibodies in plants (Hehle et al. Plant Biotechnol J. 2015; 13: 235-245). At large, the plant proteolytic machinery affects the production of many recombinant proteins. To overcome protease interference in molecular pharming, several strategies have been adopted, including overexpressing protease inhibitors or knocking down genes encoding for proteases. Co-expression of protease inhibitors along with the protein of interest either protects the recombinant protein from proteolysis or leads to increased accumulation of the product through other mechanisms. For example, SICDI, a protease inhibitor from tomato, when co-expressed, showed stabilisation of human antichymotrypsin in potato leaves [(Goulet et al. Plant Biotechnol J. 2010;8: 142-154)]. Another study reported that the transient co-expression of SICYS8 (a protease inhibitor from tomato) in N. benthamiana results in higher accumulation of C5-1 antibody [(Sainsbury et al. Plant Biotechnol J. 2013; 1 1 : 1058-1068)].

The strategy of co-expressing protease inhibitors to boost RP accumulation is known and has been successfully applied by others (Goulet et al., 2012; Jutras et al., 2016). In particular, SICYS8 is a known protease inhibitor which has been applied in plants to successfully boost RP production.

SUMMARY OF THE INVENTION

In seeking to overcome various practical disadvantages associated with the existing systems available for recombinant protein (RP) production in plants, the inventors searched for and selected, cloned and tested 29 putative candidate protease inhibitors to determine whether their co-expression boosted RP accumulation in plants. 26 of these did not boost RP accumulation, even when directed to the apoplast. Surprisingly, 3 of the tested putative protease inhibitors did boost RP accumulation on co-expression with the RP when directed to the apoplast of the host plants by fusion to the signal peptide (SP) of the NtPR1 gene.

Accordingly, the inventors provide three protease inhibitors (Pis) (HsTIMP2; NbPR4 and NbPotl) for application as tools for increasing recombinant protein (RP) expression or levels in plants. The inventors have discovered three Pis that are particularly effective at increasing protein accumulation levels of RPs when transiently co-expressed in Nicotiana benthamiana by agroinfiltration. HsTIMP2; NbPR4 and NbPotl find application as improved tools for enhancing the production of RPs in plants. All three protease inhibitors have been found to have broad application and can increase the production of a range of unrelated RPs (for example EPO, VCR01 (Light Chain and Heavy Chain), a-Gal) when co-expressed with them in host plant systems, i.e. the HIV-neutralizing antibody VRC01 , an a-galactosidase (aGal) used to treat Fabry's disease and the angiogenic glycohormone erythropoietin served as model RP to demonstrate broad functionality of each protease inhibitor (independently and in concert) with a range of different recombinant proteins. Additionally, the three protease inhibitors (HsTIMP2; NbPR4 and NbPotl) can each function alone to improve RP levels in plants or alternatively in any combination as part of a multiplex engineering system for further improving RP levels in plants.

The first of the protease inhibitors, HsTIMP2 is a human metalloprotease inhibitor that is being investigated as a potential anti-cancer agent (Wingfield et al., 1999; Arkadash et al., 2017). HsTIMP2 has never been expressed in plants, so the application of NtPR1 SP-HsTIMP2 to boost recombinant protein accumulation upon transient co-expression in N. benthamiana is extremely surprising. Additionally, the levels of RP accumulation achieved by use of HsTIMP2 are superior when compared to the best available inhibitor used so far in this platform (SICYS8).

The second of the protease inhibitors, NbPR4 is a homolog of CaPR4, which was recently shown to be a protease inhibitor (Kim & Hwang, 2015). When expressed in plants with its native signal peptide, NbPR4 co-expression did not boost RP accumulation. However, in an unexpected finding, the inventors discovered that when they redirected its expression via the secretory pathway to the apoplast of the host plant, by replacement of the native signal peptide (SP) with that of the NtPR1 gene, NbPR4 co-expression did boost RP accumulation, so the application of the NtPR1SP-NbPR4 fusion to boost recombinant protein accumulation upon transient co-expression is extremely surprising. Additionally, the levels of RP accumulation achieved by use of NbPR4 are superior when compared to the best available inhibitor used so far in this platform (SICYS8). The third of the protease inhibitors, NbPotl , was not known previously to be a protease inhibitor and nor was it linked to transient protein expression. Protein sequence analysis by the inventors indicated that it is a putative serine and cysteine protease inhibitor (MEROPS family 113) from N. benthamiana. The inventors tested two other putative 113 protease inhibitors and five more putative Ser and Cys protease inhibitors (MEROPS family I3) by generating a series of NtPR1 fusions, none of which boosted RP accumulation. However, unexpectedly, by changing the subcellular localisation of NbPotl , to generate secretory expression of NbPotl , the inventors were able to achieve an increase in recombinant protein accumulation in plants upon transient co-expression. NbPotl increases accumulation of recombinant proteins to a similar extent as the best available inhibitor used so far in this platform, SICYS8.

Accordingly, the present invention provides;

A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 1 [HsTIMP2 AA + NtPtPRISP AA] or a sequence of at least 63% identity thereto or a functional fragment thereof.

A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 26 [NbPR4 AA + NtPtPRISP AA] or a sequence of at least 63% identity thereto or a functional fragment thereof.

A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 27 [NbPotl AA + NtPtPRISP AA] or a sequence of at least 50% identity thereto or a functional fragment thereof.

Each of the protease inhibitors is capable of improving the levels of RP accumulation in a host plant or plant part upon co-expression with the RP in the host plant or plant part, compared to that of a control plant. Increasing the level of one or more recombinant proteins in a plant or plant part, in accordance with the present invention, may usefully refer to an increase in the total levels of the one or more RPs in the plant compared with an equivalent control plant. That is, as compared to the total levels of the one or more RPs (or the secretory levels of RPs) in a native (i.e. control) plant or plant part of the same species at the same stage if grown under identical conditions, which has been modified to express the relevant RP but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part; i.e. those plants in which no deliberate alteration of HsTIMP2, NbPR4 or NbPotl expression levels has been made. The protease inhibitors disclosed herein are capable of expression in the apoplast of a recipient plant. The use of these three Pis for the production of RPs in plants is a completely new contribution to the art. In particular, the inventors have discovered that expression of these Pis in the apoplast of the host plant system, via the secretory pathway, significantly boosts RP accumulation. Specifically, removal of the native signal peptides (for all three inhibitors) and fusion to NtPRI SP results in a dramatic increase in RP accumulation. HsTIMP2 has never been expressed in plants before. NbPotl has not been cloned and/or studied, as there is no record of this protein or any protein with >60% identical amino acids in the NCBI sequence database. NbPR4 and HsTIMP2 boost RP accumulation more than the best published inhibitor, SICYS8. All three inhibitors differ from SICYS8 in their putative mode of action, as they do not alter cysteine protease activity like SICYS8 does.

Increasing RP levels by co-expression of HsTIMP2

Accordingly, the present invention provides a method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide in the apoplast of the plant or plant part.

Apoplast expression (PI)

Usefully, the protease inhibitors disclosed herein are capable of expression in the apoplast of a recipient plant.

This expression may refer to over-expression, and/or spatial mis-expression of HsTIMP2 and/or NbPR4 and/or NbPotl polypeptide in a plant or plant part and/or elevation in the biological activity of the protein compared with that in an unmodified control plant. This may be achieved by various standard techniques well known in the art. In order for the protease inhibitors to have an effect on RP accumulation, HsTIMP2 and/or NbPR4 and/or NbPotl are expressed in the host plant at the same time as the relevant RP is expressed in the host plant. Co-expression of the PI and RP, which is important for increasing RP levels in host plants, typically refers to simultaneous or coincidental expression of the protease inhibitor and the recombinant protein in the plant whether temporally and/or spatially. Preferably, this is achieved by presence of a signal peptide directing translation of the protease inhibitors into the secretory pathway e.g. NtPrl signal peptide [SEQ ID NO: 28]. Thus, the Pis end up in the apoplast, but also accompany the RP throughout the secretory pathway. Preferably, the levels of PI localised in the apoplast of the recipient plant or plant part are increased, as compared to the levels in the same tissue in a native (i.e. WT control) plant or plant part of the same species at the same stage if grown under identical conditions, and in which no deliberate alteration of HsTIMP2, NbPR4 or NbPotl expression levels has been made. Preferably, the levels of HsTIMP2 and/or NbPR4 and/or NbPotl are increased. Preferably, the overall levels of HsTIMP2 and/or NbPR4 and/or NbPotl , are increased. The overall levels of HsTIMP2 and/or NbPR4 and/or NbPotl in the host plant may increase in the range 5 fold to 1000 fold relative to control plants; optionally at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold or 1000 fold that of control plants. Optionally, the overall levels of HsTIMP2 and/or NbPR4 and/or NbPotl in the host plant may increase in the range 10 fold to 20 fold, 20 fold to 30 fold, 30 fold to 40 fold, 40 fold to 50 fold, 50 fold to 60 fold, 60 fold to 70 fold, 70 fold to 80 fold, 80 fold to 90 fold or 90 fold to 100 fold relative to control plants.

In accordance with the invention, the HsTIMP2 polypeptide may comprise a polypeptide sequence of SEQ ID NO: 2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof.

Preferably, the HsTIMP2 polypeptide comprises a signal peptide capable of directing HsTIMP2 polypeptide accumulation in the apoplast of a plant. Suitably, the HsTIMP2 polypeptide may comprise a polypeptide sequence of SEQ ID NO: [HsTIMP2 AA + NtPtPRI aSP].

Preferably, the HsTIMP2 polypeptide or fragment comprises the amino acid sequence motif CSCSP [SEQ ID NO: 3]; and/or the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP [SEQ ID NO: 6]; and/or the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]; and/or the amino acid motif CSCSPVHPQQAFCNADVVIRAKAVSEKE

[SEQ ID NO: 8].

An intact N-terminus is important for function of HsTIMP2. It is important for increasing RP expression in plants that HsTIMP2 polypeptide comprises a cysteine residue at position 1 and 3 of the N-terminal amino acid motif CSCSP [SEQ ID NO: 3]. However, it is also important that the HsTIMP2 polypeptide motif is not ACSCSP. Thus the HsTIMP2 polypeptide comprises an intact N-terminus, preferably with the sequence of CSCSP. Preferably, the HsTIMP2 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof. In the case of polynucleotides encoding HsTIMP2, such polynucleotides may be derived from Homo sapiens or may be derived from any other organism.

Preferably, the present invention provides a method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide in the apoplast of the plant or plant part; and wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof; or SEQ ID NO: 10 [HsTIMP2 NT + NtPtPRISP]. Typically, said polynucleotides will be provided in an expression vector. Usually, such expression vector will be an Agrobacterium vector.

Advantageously, further increases in the level of recombinant proteins have been observed by co-expressing combinations of the protease inhibitors with the desired RPs in plants. Accordingly, in preferred aspects the present invention provides a method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and HsTIMP2 polypeptide in the apoplast of the plant or plant part; and wherein the method further comprises increasing the level or expression of NbPR4 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

The NbPR4 polypeptide may comprise; the amino acid motif TVRIVDQC [SEQ ID NO: 1 1]; and/or the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or the amino acid motif AGGQSATNVRSTYHLYNPQNINWDL [SEQ ID NO: 15]; and/or the polypeptide sequence of SEQ ID NO: 16 [NbPR4 AA] or a sequence of at least 87% identity thereto or functional fragment thereof. Most preferably, the NbPR4 polypeptide comprises all of the above amino acid motifs.

Preferably, the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof; or SEQ ID NO: 18 [NbPR4 NT + NtPtPRISP]. Advantageously, further increases in the level of recombinant proteins may be obtained by co- expressing combinations of the protease inhibitors with the desired RPs in plants. That is any combination of the protease inhibitor may be used e.g. HsTIMP2 and NbPR4, HsTIMP and NbPotl , NbPR4 and NbPotl , or HsTIMP and NbPR4 and NbPotl .

Accordingly, the method optionally further comprises increasing the level or expression of NbPotl polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

Preferably, the NbPotl polypeptide comprises; the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21 ]; and/or the amino acid motif GMPGKTAKEI IEKENPLV [SEQ ID NO: 22]; and/or the polypeptide sequence of [NbPotl AA] SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

Preferably, the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof; or SEQ ID NO: 25 [NbPotl NT + NtPtPRI SP].

Preferably, said plant or plant part is transformed with a multiplicity of said polynucleotides. Preferably, the plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, the plant or plant part has one or more of said polynucleotides stably incorporated into its genome.

Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

Preferably, the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal). Preferably, the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Optionally, the polynucleotides encoding the HsTIMP2 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins may be comprised in the same expression vector. Optionally, where HSTIMP2 is used in combination with NbPR4 and/or NbPotl to increase RP production in plants, and the method comprises transforming a recipient plant or plant part with polynucleotides encoding the one or more protease inhibitors, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and one or more polynucleotides encoding the one or more recombinant proteins may conveniently be comprised in the same expression vector.

Preferably, the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant. Preferably, the modified plant or plant part is of the genus Nicotiana, for example N. benthamiana. Preferably, said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

The present invention also provides a modified plant or plant part genetically engineered to be capable of expressing Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide in the apoplast of the plant or plant part.

Preferably, the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: 2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof. Preferably, the HsTIMP2 polypeptide comprises a signal peptide capable of directing HsTIMP2 polypeptide accumulation in the apoplast of a plant. Preferably, the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: [HsTIMP2 AA + NtPtPRI SP].

Preferably, the HsTIMP2 polypeptide comprises; the amino acid motif CSCSP [SEQ ID NO: 3]; and/or the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]; and/or the amino acid motif CSCSPVHPQQAFCNADVVIRAKAVSEKE

[SEQ ID NO: 8]. An intact N-terminus is important for function of HsTIMP2. It is important for increasing RP expression in plants that HsTIMP2 polypeptide comprises a cysteine residue at position 1 and 3 of the N-terminal amino acid motif CSCSP [SEQ ID NO: 3]. However, it is also important that the HsTIMP2 polypeptide motif is not ACSCSP. Thus the HsTIMP2 polypeptide comprises an intact N-terminus, preferably with the sequence of CSCSP.

Preferably, the HsTIMP2 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof. Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof; or SEQ I D NO: 10 [HsTIMP2 NT + NtPtPRI SP].

Preferably, the modified plant or plant part is also genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part. Preferably, the NbPR4 polypeptide comprises; the amino acid motif TVRIVDQC [SEQ I D NO: 1 1]; and/or the amino acid motif NGGLDLD [SEQ I D NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ I D NO: 13]; and/or the amino acid motif LDTNGVGYQQGHLIVNYEFI NCDD [SEQ ID NO: 14]; and/or the amino acid motif AGGQSATNVRSTYHLYNPQN I N WDL [SEQ ID NO: 15]; and/or the polypeptide sequence of SEQ ID NO: 16 [NbPR4 AA] or a sequence of at least 87% identity thereto or a functional fragment thereof.

Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof; or SEQ ID NO: 18 [NbPR4 NT + NtPtPRI SP].

Preferably, the modified plant or plant part is also genetically engineered to be capable of expressing NbPotl polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPotl polypeptide comprises; the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21 ]; and/or the amino acid motif GMPGKTAKEI IEKENPLV [SEQ ID NO: 22]; and/or the polypeptide sequence of [NbPotl AA] SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof. Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 24 [NbPotI NT] or a sequence of at least 60% identity thereto or a functional fragment thereof; or SEQ ID NO: 25 [NbPotI NT + NtPtPRISP].

Preferably, said modified plant or plant part is transformed with a multiplicity of said polynucleotides.

Preferably, said modified plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, said modified plant or plant part has one or more of said polynucleotides stably incorporated into its genome. Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

Preferably, the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of any of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part. Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3- galactose (a-Gal). Preferably, said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression any of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part.

Preferably, the polynucleotides encoding the HsTIMP2 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. Preferably, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant.

Preferably, the modified plant or plant part is of the genus Nicotiana, for example N. benthamiana. Preferably, said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells. The present invention also provides the use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: 2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof or a sequence of SEQ ID NO: 1 [HsTIMP2 AA + NtPtPRI SP AA] or a sequence of at least 63% identity thereto or a functional fragment thereof.

Increasing RP levels by co-expression of NbPR4

The present invention also provides a method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and NbPR4 polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 16 [NbPR4 AA] or a sequence of at least 87% identity thereto or a functional fragment thereof. Preferably, the NbPR4 polypeptide comprises a signal peptide capable of directing NbPR4 polypeptide accumulation in the apoplast of a plant. Preferably, the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 26 [NBPR4 AA + NtPtPRI SP]. Preferably, the NbPR4 polypeptide comprises; the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]. Preferably, the NbPR4 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof.

Preferably, the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof; or SEQ ID NO: 18 [NbPR4 NT + NtPtPRISP].

Preferably, the method further comprises increasing the level or expression of HsTIMP2 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. Preferably, the HsTIMP2 polypeptide comprises; the amino acid motif CSCSP [SEQ ID NO: 3]; and/or the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP [SEQ ID NO: 6]; and/or the amino acid motif PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP [SEQ ID NO: 7]; and/or the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE

[SEQ ID NO: 8]; and/or a polypeptide sequence of SEQ ID NO: 2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof.

An intact N-terminus is important for function of HsTIMP2. It is important for increasing RP expression in plants that HsTIMP2 polypeptide comprises a cysteine residue at position 1 and 3 of the N-terminal amino acid motif CSCSP [SEQ ID NO: 3]. However, it is also important that the HsTIMP2 polypeptide motif is not ACSCSP. Thus the HsTIMP2 polypeptide comprises an intact N-terminus, preferably with the sequence of CSCSP.

Preferably, the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof; or SEQ ID NO: 10 [HsTIMP2 NT + NtPtPRISP].

Preferably, the method further comprises increasing the level or expression of NbPotl polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

Preferably, the NbPotl polypeptide comprises; the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]; and/or the polypeptide sequence of [NbPotl AA] SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

Preferably, said plant or plant part is transformed with a multiplicity of said polynucleotides.

Preferably, the plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, the plant or plant part has one or more of said polynucleotides stably incorporated into its genome. Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

Preferably, the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal). Preferably, the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Preferably, the polynucleotides encoding the NbPR4 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the plant or plant part is of the genus Nicotiana. for example a N. benthamiana plant or plant part. Preferably, said plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

The present invention also provides a modified plant or plant part genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 16

[NbPR4 AA] or a sequence of at least 87% identity thereto or a functional fragment thereof.

Preferably, the NbPR4 polypeptide comprises a signal peptide capable of directing NbPR4 polypeptide accumulation in the apoplast of a plant. Preferably, the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 26 [NbPR4 AA + NtPtPRISP]. Preferably, the NbPR4 polypeptide comprises; the amino acid motif TVRIVDQC [SEQ ID NO: 1 1]; and/or the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or the amino acid motif AGGQSATNVRSTYHLYNPQNINWDL [SEQ ID NO: 15].

Preferably, the NbPR4 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof.

Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof; or SEQ ID NO: 18 [NbPR4 NT + NtPtPRISP].

Preferably, the modified plant or plant part is genetically engineered to be capable of expressing HsTIMP2 polypeptide in the apoplast of the plant or plant part.

Preferably, wherein the HsTIMP2 polypeptide comprises; the amino acid motif CSCSP [SEQ ID NO: 3]; and/or the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or

the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]; and/or the amino acid motif CSCSPVH PQQAFCNADVVI RAKAVSEKE

Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof; or SEQ ID NO: 10 [HsTIMP2 NT + NtPtPRISP]. Preferably, the modified plant or plant part is genetically engineered to be capable of expressing NbPotl polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPotl polypeptide comprises; the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21 ]; and/or the amino acid motif GMPGKTAKEI IEKENPLV [SEQ ID NO: 22]; and/or the polypeptide sequence [NbPotl A A] [SEQ ID NO: 23] or a sequence of at least 50% identity thereto or a functional fragment thereof.

Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof; or SEQ ID NO: 25 [NbPotl NT + NtPtPRI SP].

Preferably, said modified plant or plant part is transformed with a multiplicity of said polynucleotides. Preferably, said modified plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, said modified plant or plant part has one or more of said polynucleotides stably incorporated into its genome. Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

Preferably, the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3- galactose (a-Gal). Preferably, said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Preferably, the polynucleotides encoding the NbPR4 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. Preferably, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant. Preferably, the modified plant or plant part is of the genus Nicotiana for example N. benthamiana. Preferably, said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

The present invention also provides the use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: 16 [NbPR4 AA] or a sequence of at least 87% identity thereto or a functional fragment thereof or a sequence of SEQ ID NO: 26 [NbPR4 AA + NtPtPRI SP AA] or a sequence of at least 63% identity thereto or a functional fragment thereof.

Increasing RP levels by co-expression of NbPotI

The present invention also provides a method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and NbPotI polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 23 [NbPotI AA] or a sequence of at least 50% identity thereto or a functional fragment thereof. Preferably, the NbPotI polypeptide comprises a signal peptide capable of directing NbPotI polypeptide accumulation in the apoplast of a plant. Preferably, the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 27 [NBPotl AA + NtPtPRI aSP].

Preferably, the NbPotI polypeptide comprises;

the amino acid motif KXiX₂WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]. Preferably, the NbPotl polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof.

Preferably, the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof; or SEQ ID NO: 25 [NbPotl NT + NtPtPRI SP].

Preferably, the method further comprises increasing the level or expression of HsTIMP2 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]; and/or the amino acid motif CSCSPVHPQQAFCNADVVIRAKAVSEKE

Preferably, the method further comprises increasing the level or expression of NbPR4 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. Preferably, the NbPR4 polypeptide comprises; the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or the amino acid motif

LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or the amino acid motif

AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]; and/or the polypeptide sequence of [NbPR4 AA] SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof.

Preferably, the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; SEQ ID NO: 17 [NbPR4 NT] or a sequence of at least 72% identity thereto or a functional fragment thereof; or SEQ ID NO: 18 [NbPR4 NT + NtPtPRISP].

Preferably, said plant or plant part is transformed with a multiplicity of said polynucleotides. Preferably, the plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, the plant or plant part has one or more of said polynucleotides stably incorporated into its genome. Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

Preferably, the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal). Preferably, the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

Preferably, the polynucleotides encoding the NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. Preferably, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. Preferably, the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant. Preferably, the plant or plant part is of the genus Nicotiana for example N. benthamiana. Preferably, said plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

The present invention also provides a modified plant or plant part genetically engineered to be capable of expressing NbPotl polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPotl polypeptide comprises a polypeptide sequence of SEQ ID NO: 23 [NbPotl AA] or a sequence of at least 50% identity thereto or a functional fragment thereof.

Preferably, the NbPotl polypeptide comprises a signal peptide capable of directing NbPotl polypeptide accumulation in the apoplast of a plant. Preferably, the NbPotl polypeptide comprises a polypeptide sequence of SEQ ID NO: 27 [NbPotl AA + NtPtPRISP].

Preferably, the NbPotl polypeptide comprises; the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22].

Preferably, the NbPotl polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof. Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 24 [NbPotl NT] or a sequence of at least 60% identity thereto or a functional fragment thereof; or SEQ ID NO: 25 [NbPotl NT + NtPtPRISP].

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]; and/or the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO: 8]; and/or a polypeptide sequence of SEQ ID NO: 2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof.

Preferably, the modified plant or plant part is transformed with a polynucleotide comprising; SEQ ID NO: 9 [HsTIMP2 NT] or a sequence of at least 64% identity thereto or a functional fragment thereof; or SEQ ID NO: 10 [HsTIMP2 NT + NtPtPRISP].

Preferably, the modified plant or plant part is genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part.

Preferably, the NbPR4 polypeptide comprises; the amino acid motif TVRIVDQC [SEQ ID NO: 1 1]; and/or the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or the amino acid motif AGGQSATNVRSTYHLYNPQNINWDL [SEQ ID NO: 15]; and/or the polypeptide sequence of [NbPR4 AA] SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof.

Preferably, said modified plant or plant part is transformed with a multiplicity of said polynucleotides. Preferably, said modified plant or plant part is transiently transformed with one or more of said polynucleotides. Alternatively, said modified plant or plant part has one or more of said polynucleotides stably incorporated into its genome.

Preferably, one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part. Preferably, the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part. Preferably, the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal). Preferably, said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part.

Preferably, the polynucleotides encoding the NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

Preferably, the modified plant or plant part is of the genus Nicotiana, for example N. benthamiana. Preferably, said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

The present invention also provides the use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: 23 [NbPotI AA] or a sequence of at least 50% identity thereto or a functional fragment thereof; or a sequence of SEQ ID NO: 27 [NbPotI AA + NtPtPRI SP AA] or a sequence of at least 50% identity thereto or a functional fragment thereof.

Polynucleotides

Throughout, polynucleotides encoding the protease inhibitors HsTIMP2, NbPR4, and NbPotI may be isolated nucleic acid molecules and may be RNA or DNA molecules. Throughout, the term "polynucleotide" as used herein refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form, or sense or anti-sense, and encompasses analogues of naturally occurring nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. In the case of polynucleotides encoding NbPR4, and NbPotl , such polynucleotides may be derived from Nicotiana sp. or may be derived from any other plant. In the case of polynucleotides encoding HsTIMP2, such polynucleotides may be derived from Homo sapiens or may be derived from any other organism.

In all aspects of the invention which comprise transforming the plant or plant part with a polynucleotide encoding HsTIMP2 or NbPR4 or NbPotl , the recipient plant or plant part may be transformed with a multiplicity of said polynucleotides. The recipient plant or plant part may be transiently transformed with one or more of said polynucleotides. The recipient plant or plant part may have one or more of said polynucleotides stably incorporated into its genome. Preferably, one or more of the polynucleotides may be expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide (i.e. HsTIMP2 or NbPR4 or NbPotl) compared to that of an equivalent untransformed control plant or plant part.

Polypeptide and Polynucleotide Sequence Identity

Throughout, the invention provides modified protease inhibitors having reference polypeptide or polynucleotide sequences, but it will be appreciated that these reference sequences include any variant sequence having the defined percentage identity therewith. Such percentage identities include any of the following: where a reference nucleic acid or polypeptide sequence and sequences of at least a certain percentage identity are disclosed, e.g. at least 50%, then optionally the percentage identity may be different. For example: a percentage identity which is selected from one of the following: at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.7% or at least 99.8%. Such sequence identity with an amino acid or nucleic acid sequence is a function of the number of identical positions shared by the sequences in a selected comparison window, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of polypeptides, proteins, polynucleotides (comprising RNA, DNA or synthetic nucleic acids) in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet.

In all aforementioned aspects of the present invention, amino acid residues may be substituted conservatively or non-conservatively. Conservative amino acid substitutions refer to those where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the resulting polypeptide. Similarly, it will be appreciated by the skilled reader that nucleic acid sequences may be substituted conservatively or non-conservatively without affecting the function of the polypeptide. Conservatively modified nucleic acids are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences. It will be appreciated by the skilled reader that each codon in a nucleic acid (except AUG and UGG; typically, the only codons for methionine or tryptophan, respectively) can be modified to yield a functionally identical molecule. Accordingly, each silent variation (i.e. synonymous codon) of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.

Modulating the levels or activity of a polypeptide encoded by a nucleic acid molecule may be achieved by various means. For example, elevating mRNA levels encoding said polypeptide by placing the nucleotide under the control of a strong promoter sequence or altering the gene dosage by providing a cell with multiple copies of said gene or its complement. Alternatively, the stability of the mRNA encoding said polypeptide may be modulated to alter the steady state levels of an mRNA molecule, this is preferably achieved via alteration to the 5' or 3' untranslated regions of the mRNA. Similarly, the production of a polypeptide may be modified by altering the efficiency of translational processing, increasing or decreasing protein stability or by altering the rate of post translational modification (e.g. proteolytic cleavage) or secretion. Typically, in order to achieve higher yields of RP in host plants or plant parts, high levels of expression and/or activity of HsTIMP2 and/or NbPR4 and/or NbPotl will be desired in the apoplast of the host plants or plant parts. Preferably, the overall levels of HsTIMP2 and/or NbPR4 and/or NbPotl , are increased. The overall levels of HsTIMP2 and/or NbPR4 and/or NbPotl may increase in the range 5 fold to 1000 fold relative to control plants; optionally 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold or 1000 fold that of control plants.

Commonly, where a plant naturally expresses said gene, for example as in the case of NbPR4 and/or NbPotl in N. benthaminana, their expression in the apoplast may be achieved by altering the expression pattern of the native gene(s) and/or the processing and/or production of the polypeptide. This may be achieved by any suitable method, including, but not limited to altering transcription of the gene, and/or translation of the mRNA into polypeptide, and post- translational modification of the polypeptide. Altering the expression pattern of a native gene may be achieved by placing it under control of a heterologous regulatory sequence, which is capable of directing the desired expression pattern of the gene. Suitable regulatory sequences may be placed 5' and/or 3' of the endogenous gene and may include, but are not limited to promoter sequences, terminator fragments, polyadenylation sequences or enhancer sequences (e.g. VP16 transactivation domain) operably linked to the sequences of interest.

Plants transformed with a polynucleotide or expression construct encoding HsTIMP2, NbPR4 or NbPotl may be produced by standard techniques for the genetic manipulation of plants which are known in the art. DNA may be introduced into plant cells using any suitable technology, such as gene transfer via a disarmed Ti-plasmid vector carried by Agrobacterium tumefaciens, using Agrobacterium sp.-mediated transformation, vacuum infiltration, floral dip, spraying, particle or microprojectile bombardment, protoplast transformation, electroporation, microinjection, electrophoresis, pollen-tube pathway, silicon carbide- or liposome-mediated transformation, uptake by the roots, direct injection into the xylem or phloem or other forms of direct DNA uptake. Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium-coated micro particles or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium.

According to the present invention, whole plants, plant material or plant parts may be stably or transiently transformed as desired, wherein stable transformation refers to polynucleotides which become incorporated into the plant host chromosomes such that the host genetic material may be permanently and heritably altered and the transformed cell may continue to express traits caused by this genetic material, even after several generations of cell divisions.

Transiently transformed plant cells refer to cells which contain heterologous DNA or RNA, and are capable of expressing the trait conferred by the heterologous genetic material, without having fully incorporated that genetic material into the cell's DNA. Heterologous genetic material may be incorporated into nuclear or plastid (chloroplastic or mitochondrial) genomes as required to suit the application of the invention. Where plants are transformed with more than one polynucleotide it is envisaged that combinations of stable and transient transformations are possible. More commonly, plants or plant parts of the invention may be stably or transiently transformed with polynucleotides encoding HsTIMP2 and/or NbPR4 and/or NbPotl Preferably, plants or plant parts of the invention are will be transformed with polynucleotides encoding HsTIMP2 and/or NbPR4 and/or NbPotl

The polypeptide sequences and polynucleotides used in the present invention may be isolated or purified. By "purified" is meant that they are substantially free from other cellular components or material, or culture medium. "Isolated" means that they may also be free of naturally occurring sequences which flank the native sequence, for example in the case of nucleic acid molecule, isolated may mean that it is free of 5' and 3' regulatory sequences. According to the present invention, for use in expressing the HsTIMP2 or NbPR4 or NbPotl in the apoplast of a plant, polynucleotide sequences may be integrated into an expression cassette comprising a regulatory sequence to express the relevant HsTIMP2 or NbPR4 or NbPotl genes in the apoplast of a plant or plant part. Alternatively, for use in expressing the HsTIMP2 or NbPR4 or NbPotl in the apoplast of a plant or plant part, polynucleotide sequences may be integrated into an expression cassette comprising a regulatory sequence to express the relevant HsTIMP2 or NbPR4 or NbPotl genes in the cells of a plant and subsequently be transported to the apoplast of a plant or plant part. Preferably, the regulatory sequences are designed to be operably linked to the relevant polynucleotides, in order to direct expression in a manner according to the present invention.

Expression Cassettes

The polynucleotides as described herein (namely those encoding HsTIMP2 or NbPR4 or

NbPotl) and/or one or more regulatory sequences are preferably provided as part of an expression cassette for expression of the polynucleotide in a cell or tissue of interest. In some instances, the expression cassettes will also comprise polynucleotides sequences encoding the desired RP (for example EPO, VCR01 or a-Gal). Suitable expression cassettes for use in the present invention may be constructed by standard techniques known in the art, to comprise

5' and 3' regulatory sequences, including, but not limited to promoter sequences, terminator fragments, polyadenylation sequences or enhancer sequences (e.g. VP16 transactivation domain) operably linked to the sequences of interest. Such elements may be included in the expression construct to obtain the optimal expression and function of HsTIMP2 or NbPR4 or

NbPotl in the plant or plant part. In addition, polynucleotides encoding, for example, selectable markers and reporter genes may be included. The expression cassette preferably also contains one or more restriction sites, to enable insertion of the nucleotide sequence and/or a regulatory sequence into the plant genome, at pre-selected loci. Also provided on the expression cassette may be transcription and translation initiation regions, to enable expression of the incoming genes, transcription and translational termination regions, and regulatory sequences. These sequences may be native to the plant being transformed, or may be heterologous. The expression cassettes may be a bi-functional expression cassette which functions in multiple hosts.

Regulatory Sequences

A regulatory sequence is a nucleotide sequence which is capable of influencing transcription or translation of a gene or gene product, for example in terms of initiation, accuracy, rate, stability, downstream processing and mobility. Examples of regulatory sequences include promoters, 5' and 3' UTR's, enhancers, transcription factor or protein binding sequences, start sites and termination sequences, ribosome binding sites, recombination sites, polyadenylation sequences, sense or antisense sequences. They may be DNA, RNA or protein. The regulatory sequences may be plant-, bacteria-, fungal- or virus derived, and preferably may be derived from the same species of plant as the plant being modulated.

Promoters

It will be appreciated that a variety of promoters are suitable for use with the invention. Accordingly, depending on the desired application and particular arrangement, suitable promoters may be constitutive, whereby they direct expression under most environmental conditions or developmental stages, developmental stage specific, tissue-specific or inducible (e.g. to direct expression in response to environmental, chemical or developmental cues, such as temperature, light, chemicals, drought, and other stimuli). Typically, the promoters controlling the expression of HsTIMP2 and/or NbPR4 and/or NbPotl will be constitutive, such that high-levels of expression of HsTIMP2 and/or NbPR4 and/or NbPotl are achieved in the host plant, for example the CaMV35S promoter, although many other suitable promoters will be known to the skilled person. To improve RP levels in plants, the accumulation of the one or more protease inhibitors is required in the secretory pathway, however, when transient expression systems, such as via Agrobacterium are used, promoters which are not tissue specific may be used, and expression restricted to the agroinfiltrated tissues anyway, since Agrobacteria do not move through the plant. In this way, strong expression of each of the Pis may be achieved and high-levels of Pis can be allowed to accumulate in the secretory pathway. Preferably, therefore, the promoter is a constitutive promoter, e.g. the CaMV 35S promoter. In some instances it may be desired to combine the strong expression of a constitutive promoter and rely on a signal peptide to localise the protease inhibitor to the secretory pathway and eventually, the apoplast, for example the NtPR1 signal peptide.

Suitable promoter sequences may include but are not limited to those of the T-DNA of A tumefaciens, including mannopine synthase, nopaline synthase, and octopine synthase; alcohol dehydrogenase promoter from Zea mays; light inducible promoters such as ribulose- biphosphate-carboxylase small subunit gene from various species and the major chlorophyll a/b binding protein gene promoter; histone promoters, actin promoters; Zea mays ubiquitin 1 promoter; 35S and 19S promoters of cauliflower mosaic virus; developmental^ regulated promoters such as the waxy, zein, or bronze promoters from Zea mays; as well as synthetic or other natural promoters including those promoters capable of driving high-levels of transcript accumulation in Nicotiana benthamiana plants. These promoters may be derived from Nicotiana benthamiana or any other suitable organism.

Secretory Pathway Signal Peptide

Polynucleotides encoding HsTIMP2 and/or NbPR4 and/or NbPotl will preferably be provided in an expression vector or expression cassette. The expression cassette comprising the polynucleotide encoding HsTIMP2 and/or NbPR4 and/or NbPotl may also comprise sequences coding for a transit or signal peptide, to drive the protein encoded by the heterologous polynucleotide into the secretory pathway and eventually the apoplast. This ensures co-expression of the PI and RP, which is important for increasing RP levels in host plants. The signal peptide ensures translation of the protease inhibitors into the secretory pathway. Thus, the Pis end up in the apoplast, but also accompany the RP throughout the secretory pathway. Whilst not wishing to be bound by any particular theory, the inventors suspect this co-secretion of PI and RP may be important, as proteolytic degradation likely happens in the secretory pathway. Therefore it is preferable that any signal peptide used in accordance with the invention is capable of directing the translation of the protease inhibitors into the secretory pathway. Such transit peptides are well known to those of ordinary skill in the art, and may include single transit peptides, as well as multiple transit peptides obtained by the combination of sequences coding for at least two transit peptides. Preferably, the expression cassette comprising the polynucleotide encoding the HsTIMP2 and/or NbPR4 and/or NbPotl polypeptide, comprises a signal peptide capable of directing HsTIMP2 and/or NbPR4 and/or NbPotl polypeptide accumulation in the apoplast of a plant. Preferably, the signal peptide comprises a polypeptide sequence of

MGFVLFSQLPSFLLVSTLLLFLVISHSCRAGG [SEQ ID NO: 28]. Preferably the signal peptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 29 [NtPtPRI SP NT]. Subsequently, in some instances, the expression levels of the HsTIMP2 and/or NbPR4 and/or NbPotl protein in host organisms of interest may be determined. In some instances, it may be possible to directly determine functional expression, e.g. as with GFP or by enzymatic action of the protein of interest (POI) to generate a detectable optical signal. However, in some instances it may be chosen to determine physical expression, e.g. by antibody probing, and rely on separate test to verify that physical expression is accompanied by the required function.

In some instances it may be desirable that HsTIMP2 and/or NbPR4 and/or NbPotl expression in host plant or plant part will be detectable by a high-throughput screening method, for example, relying on detection of an optical signal. For this purpose, it may be necessary for the protein of interest (POI) to incorporate a tag, or be labelled with a removable tag, which permits detection of expression. Where the POI is a protein to be used as a therapeutic, any tag employed for detection of expression may be cleavable from the POI. Other kinds of label may be used to mark the nucleic acid including organic dye molecules, radiolabels and spin labels which may be small molecules. However, whilst visualisation might be desirable, this is not critical to the application of the invention. Indeed, the inventors have found that for each of the protease inhibitors (HsTIMP2, NbPR4 and NbPotl) the presence of intact N- and C- termini is important in achieving high levels of RP accumulation in host plants. Incorporation of some tags (e.g. FLAG) can prevent accumulation of RP. Therefore any tag or means of visualisation used in accordance with the invention must allow the N- and/or C-terminus of the protease inhibitor to remain intact and functional.

Expression Vector Delivery

The expression vectors comprising polynucleotides encoding HsTIMP2 or NbPR4 or NbPotl polypeptides can be delivered in several different ways into the recipient plant, such as for example, Nicotiana benthamiana.

Optionally, the vectors may be delivered into the recipient plant by methods which are well known in the art, for example direct agro-inoculation of Agrobacterium cultures transformed with binary modified expression vectors. Alternatively, the vectors may be delivered into the recipient plant by rub-inoculation of plant leaves with in vitro transcript. In another alternative, the vectors may be delivered into the recipient plant by bombardment of plant leaves with e.g. gold particles coated with the plasmids. Infection of the plants in accordance with the methods of the invention may involve a single application of the vector to the plant. However, it will be understood that treatment may alternatively involve multiple applications of the same vector or composition or indeed combinations of the modified expression vectors disclosed herein. Where multiple (i.e. two or more) different vectors are applied to the same plant, these may be applied simultaneously, separately (in any order) or sequentially. The vectors may be delivered into the recipient plant by any combination of the above methods.

Host Plants

In all aspects of the present invention, plants, plant material or plant parts may refer to any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

According to all aspects of the present invention, any dicotyledonous plant species may be used as a host system for co-expression of HsTIMP2, and/or NbPR4 and/or NbPotl alone, or in combination with a recombinant protein which is desired for expression. In accordance with all aspects of the invention the host or recipient plant may be a dicotyledonous plant, or the plant part is of a dicotyledonous plant. Typically, the plant or plant part is of the genus Nicotiana, for example N. benthamiana.

Preferred plants for use in the present invention are those which are genetically tractable and are typically exploited for protein production, exhibit high growth rates, are easily grown, are easily harvested, and from which recombinant proteins can readily be harvested and purified. In particular, plant transient expression systems, with eukaryotic post-translational modification machinery, offer superior efficiency, scalability, safety, and lower cost over other expression systems. However, it will be appreciated that due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, depending on the RP it may in certain circumstances preferable to use a host system produce target RPs in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Preferred plants include those for which well-developed systems for RP production exist Nicotiana tabacum and Nicotiana benthamiana and more particularly those varieties or lines which have been engineered to produce a non-glycosylated RPs, which are well known in the art, for example ΔΧΤ/FT plants as described in Castilho, A., Wndwarder, M., Gattinger, P., Mach, L, Strasser, R., Altmann, F. and Steinkellner, H. (2014) Proteolytic and N-glycan processing of human a1- antitrypsin expressed in Nicotiana benthamiana. Plant Physiol. 166, 1839-1851. Further glyco-engineered lines are described in Schoberer J, Strasser R. 2017. Plant glyco- biotechnology. Seminars in Cell & Developmental Biology.

Recombinant Proteins

Whilst the strategy of co-expressing protease inhibitors to boost RP accumulation is known, the protease inhibitors disclosed herein are superior in boosting RP accumulation when compared to the best available protease inhibitor in this platform (e.g. SICYS8), whether used alone or in any combination.

Advantageously, throughout, methods of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and HsTIMP2 and/or NbPR4 and/or NbPotl polypeptide in the apoplast of the plant or plant part will result in a recipient plant or plant part that has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the relevant recombinant protein(s) but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. The overall levels of RP or secretory or apoplastic levels of RP may increase in the range 2 fold to 500 fold relative to such control plants; optionally 1.5-fold to 10-fold, 10 fold to 100-fold or 100 fold to 500-fold sounds reasonable, optionally at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold or 500 fold that of such control plants. Typically, levels of RP achieved in host plants may be in the range 10-500 mg, preferably 15- 100 mg RP (e.g. antibody) per kg fresh leaf biomass, optionally at least 10, at least 20, at least 30, at least 40 at least 50, at least 60, at least 70, at least 80, at least 90 at least 100 mg RP (e.g. antibody) per kg fresh leaf biomass.

Alternatively, methods of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and HsTIMP2 and/or NbPR4 and/or NbPotl polypeptide in the apoplast of the plant or plant part will result in a recipient plant or plant part that has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to co- express the relevant recombinant protein(s) and SICYS8, but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. The overall levels of RP or secretory or apoplastic levels of RP may increase in the range 2 fold to 500 fold relative to such control plants; optionally at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 450 fold or 500 fold that of such control plants.

Usefully, each of the protease inhibitors disclosed herein is of broad application and may be used independently or in combination to improve the expression or level of a range of different recombinant proteins in plants. Preferably, the one or more recombinant proteins is VCR01 (heavy and/or light chains, HC/LC) and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3- galactose (a-Gal). Preferably, the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. Accordingly, the plant or plant part has an increased level of recombinant VCR01 (LC/HC) and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of any of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

DETAILED DESCRIPTION

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in detail with reference to examples and with reference to the accompanying drawings, in which:

Figure 1 shows an amino acid sequence alignment of CaPR4 to NbPR4.

Figure 2 shows transient Expression of NbPR4 in N. benthamiana does not cause cell death.

Figure 3 shows transient expression of NbPR4 results in higher accumulation of VRC01 when co-expressed.

Figure 4 shows NbPR4 enhances accumulation of Erythropoietin (EPO) and Alpha- galactosidase.

Figure 5 shows NbPR4 enhances accumulation of VRC01 , EPO and Alpha-Gal upon co- expression of all three-recombinant proteins.

Figure 6 shows C-Terminal FLAG tag disrupts NbPR4 functionality.

Figure 7 shows NbPR4 FLAG-tag and non-tag accumulate in the apoplastic fluid. Figure 8 shows co-expression of protease inhibitors enhances accumulation of VRC01 , EPO and Alpha-Gal.

Figure 9 shows NbPR4 does not affect cysteine protease activity profile of N. benthamiana.

Figure 10 shows NbPR4 does not affect extracellular cysteine protease activity profile of N. benthamiana.

Figure 11 shows NbPR4 does not affect serine protease activity profile of N. benthamiana.

Figure 12 shows NbPR4 does not affect extracellular serine protease activity profile of N. benthamiana.

Figure 13 shows a sequence alignment of CaPR4c to NbPR4.

Figure 14 shows Trypan blue staining of full leaf: NbPR4 does not cause cell death in N. benthamiana (full leaf picture of Figure 2).

Figure 15 shows NbPR4 does not cause cell death in N. benthamiana (without positive control)

Figure 16 shows time dependent VRC01 production in N. benthamiana.

Figure 17 shows a binary vector for NbPR4.

Figure 18 shows a binary vector for EPO.

Figure 19 shows a binary vector for VRC01 (LC) and VRC01 (HC).

Figure 20 shows a binary vector for alpha-Gal.

Figure 21 shows co-expression of NbPR4, NbPotI or HsTIMP enhances accumulation of aGal (a), EPO (b) and VRC01 (c).

Figure 22 shows combinations of NbPR4, HsTIMP and SICYS8 enhance accumulation of aGal, EPO and VRC01.

Figure 23 shows intact and mutant inhibitor proteins accumulate in N. benthamiana leaves upon transient expression.

Figure 24 shows Asp and Ser proteases are depleted upon NbPR4 and NbPotI overexpression, while proteins associated with defence signalling accumulate upon NbPR4, NbPotI , HsTIMP and SICYS8 overexpression.

Figure 25 shows in vitro proteolytic degradation of VRC01 is inhibited by CYS8.

Figure 26 shows activity-based protein profiling reveals that NbPR4, NbPotI and HsTIMP differ from SICYS8 in their mode of action.

Figure 27 shows fusion of NtPR1 signal peptide to NbPR4 enhances VRC01 accumulation. Figure 28 shows degradation of both fluorescent and unlabelled VRC01 happens in vitro and is limited by the inhibitor cocktail.

Figure 29 shows a matrix of the percentage of identical amino acids as between HsTIMP2 (protein) with homologues from the NCBI landmark database.

Figure 30 shows an alignment of HsTIMP2 (nucleotide) with homologues from the NCBI nr database.

Figure 31 shows a matrix of the percentage of identical nucleotides as between HsTIMP2 (nucleotide) with homologues from the NCBI nr database.

Figure 32 shows a matrix of the percentage of identical amino acids as between NbPR4 (protein) with homologues from the NCBI landmark database.

Figure 33 shows an alignment of NbPR4 cDNA with homologues from the NCBI nr database.

Figure 34 shows a matrix of the percentage of identical nucleotides as between NrPR4 cDNA with homologues from the NCBI nr database.

Figure 35 shows a matrix of the percentage of identical amino acids as between NbPotl (protein) with homologues from the NCBI landmark database

Figure 36 shows a matrix of the percentage of identical nucleotides as between NbPotl (nucleotide) with homologues from the NCBI nr database.

Figure 37 shows an alignment of NbPotl (protein) with the two other N. benthamiana 113 inhibitors, neither of which showed a clear increase in RP accumulation upon co-expression.

Figure 38 shows a matrix of the percentage of identical amino acids as between NbPotl (protein) with and the two other N. benthamiana 113 inhibitors, neither of which showed a clear increase in RP accumulation upon co-expression.

Figure 39 shows a matrix of the percentage of identical nucleotides as between NbPotl (nucleotide) and the two other N. benthamiana 113 inhibitors, neither of which showed a clear increase in RP accumulation upon co-expression.

Figure 40 shows an amino acid sequence alignment of all three protease inhibitors (HsTIMP2, NbPR4, NbPotl) with SICYCS8.

Figure 41 shows a matrix of the percentage of identical amino acids as between all three protease inhibitors (HsTIMP2, NbPR4, NbPotl) with SICYCS8.

Figure 42 shows an alignment of HsTIMP2 (protein) with homologues from the NCBI landmark database Figure 43 shows an alignment of HsTIMP2 (nucleotide) with homologues from the NCBI nr database and provides partial replication of information in Figure 30.

Figure 44 shows an alignment of NbPR4 (protein) with homologues from the NCBI landmark and nr databases.

Figure 45 shows an alignment of NbPR4 cDNA with homologues from the NCBI nr database and provides partial replication of information in Figure 33.

Figure 461 shows an alignment of NbPotl (protein) with homologues from the NCBI landmark database.

Figure 47 shows an alignment of NbPotl (nucleotide) with homologues from the NCBI nr database.

Figure 48 shows an amino acid sequence alignment of all three protease inhibitors (HsTIMP2, NbPR4, NbPotl) with SICYCS8 and provides partial replication of information in Figure 40.

Figure 49 shows an alignment of NbPotl (nucleotide) with nucleotide sequences of the two other N. benthamiana 113 inhibitors, neither of which showed a clear increase in RP accumulation upon co-expression.

Examples

Example 1 : Sequence alignment of CaPR4 to NbPR4

The pepper (Capsicum annuum) protein CaPR4 is a strong cysteine protease inhibitor involved in plant immunity. When transiently overexpressed with its native signal peptide, CaPR4 localises in the plasma membrane and causes cell death in pepper. Figure 1 shows an amino acid sequence alignment of CaPR4 to NbPR4. Amino acid sequence of CaPR4c and NbPR4 were aligned using pairwise align in Geneious software (local alignment-smith waterman, costmatrix: Blosum62), Which showed 86% similarity (Figure 13). Consensus sequence at the top and the green color is indicating similarity between NbPR4 and CaPR4. Signal peptides were omitted from both proteins as they are not in our (main) expression vector. The N. benthamiana genome encodes for a protein that is 86% identical to CaPR4 and was therefore named NbPR4.

Example 2. Transient Expression of NbPR4 in N. benthamiana does not cause cell death

Plants were agroinfiltrated with transformed A. tumefaciens containing vectors encoding for NbPR4- native Signal peptide and NbPR4- NtPRIa (signal peptide) at OD = 1.0 except positive control which was OD= 0.2, negative control refers to when the plant was infiltrated with infiltration buffer only. Figure 2 panel (a) shows cell-death phenotype in leaf two days after infiltration. Figure 2 panel (b) shows trypan blue was performed on the leaf (a). Figure 2 panel (c) shows cell-death phenotype four days after infiltration. Figure 2 panel (d) shows trypan blue was performed on the leaf (c).

We intended to investigate if NbPR4 causes cell death in N. benthamiana. For this, we used Agrobacterium tumefaciens bacteria in which the T-DNA binary system was modified to contain either the genes encoding for NbPR4 with its native signal peptide or the NbPR4 with an engineered signal peptide, NtPRIa. Plant leaves were agroinfiltrated with these strains. On the leaves, no cell death symptoms were visually observed after two and four days post- infiltration, with the exception of the positive control (Figure 2). The same results were observed for the trypan blue staining, where cell death also occurred in the positive control. Co-expression of the tomato Rx protein with the coat protein of potato virus x is known to cause cell death in N. benthamiana and it was used as a positive control in this experiment. In summary, NbPR4 does not cause cell death in N. benthamiana when transiently expressed.

Example 3: Transient expression of NbPR4 results in higher accumulation of VRC01 when co-expressed.

Co-expression of a protease inhibitor to protect recombinant proteins from degradation in N. benthamiana have been successfully studied previously. We have shown that NbPR4 does not cause cell death in N. benthamiana whether with its native signal peptide or with the NtPRIa signal peptide. Therefore, we wanted to use it in molecular pharming to prevent degradation of recombinant proteins expressed in N. benthamiana. We hypothesised that NbPR4 may inhibit cysteine proteases like CaPR4 does.

We have used the signal peptide NtPRIa fused with NbPR4, which targets the protein to the plant apoplast. Plant based recombinant proteins pass through the cell secretory pathway. We hypothesised that the recombinant protein will be secreted along with NbPR4-NtPR1a and will be protected from degradation.

To test if co-expression of NbPR4 results in higher yield of recombinant proteins in N. benthamiana, expression constructs for the genes encoding for NbPR4, VRC01 heavy chain + light chain (HC+LC), erythropoietin and alpha-galactosidase were designed and A. tumefaciens GV3101 was transformed with these plasmids. Leaves of N. benthamiana were infiltrated with the resulting A. tumefaciens strains to transiently express NbPR4 and VRC01 (HC+LC). Total leaf extract was harvested at 3dpi and submitted to SDS-PAGE 4-15 % under reducing and non-reducing conditions. Degradation of VRC01 was observed most clearly at 3 dpi in N. benthamiana (Figure 16). Immunoblots for the representation of "VRC01" HC+LC (heavy chain and Light chain), and NbPR4 when transiently expressed in N. benthamiana are shown in Figure 3. Full leaf extract was prepared in PBS (Phosphate-buffered saline) at pH=7.4 after 3dpi. Total leaf extract was submitted to SDS 4-15% PAGE under reducing or non-reducing conditions. For Immunoblot analysis proteins were electrotransferred onto PVDF (Polyvinylidene difluoride) membrane. Total protein was visualised by Coomassie staining. Figure 3 panel (a) shows an immunoblot for representative of full-size VRC01 , total leaf extract was run under non-reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-Human Kappa to detect light chain, positive control (+ve) refers to the purified antibody. Figure 3 panel (b) shows an immunoblot for representative of Heavy chain, total leaf extract was run under reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-Human Gamma to detect heavy chain. Visual illustration of bands is shown on right side and position of the molecular mass marker is shown at the left side (kDa). WT (Wild-type) stands for plasmid without T-DNA.

When co-expressed with NbPR4, VRC01 accumulates more compared to the control, which was infiltrated with A. tumefaciens strains for VRC01 expression and untransformed A. tumefaciens GV3101 (Figure 3). VRC01 is a monoclonal antibody, consisting of two heavy chains (100 kilodalton (kDa)) and two light chains (50kDa) which are joined by two disulphide bonds. Endogenous proteases often degrade antibodies expressed in plants. Expression of VRC01 resulted in the accumulation of the fully assembled antibody, which runs at a molecular weight of 180 kDa, at approximately the same level compared to the purified VRC01 (positive control) (Figure 3a).

In addition to the fully assembled VRC01 , we detected multiple bands that may result from proteolytic degradation. The detected band around 100 kDa probably represents the full antibody without the Fc domain. The band around 40 kDa may represent one single Fab fragment, and the band around 25kDa represents the VRC01 light chain. Under reducing conditions, the disulphide bridge between HC and LC was broken. The band detected around 50 kDa represents the VRC01 heavy chain (Figure 3b). As all bands accumulate more when VRC01 is co-expressed with NbPR4, we cannot argue that NbPR4 completely prevents its proteolytic degradation. However, co-expression of NbPR4 facilitates the accumulation of VRC01 at much higher levels compared to expression of VRC01 alone.

Example 4: NbPR4 enhances accumulation of Erythropoietin (EPO) and Alpha- galactosidase. Immunoblots for the representation of Erythropoietin (EPO) and Alpha-galactosidase (Alpha- Gal) when they are transiently expressed in plant are shown in Figure 4. Full leaf extract was prepared in PBS at pH=7.4 after 3dpi. Total leaf extract was submitted to SDS 4-15% PAGE under reducing conditions. For Immunoblot analysis proteins were electrotransferred onto PVDF membrane and probed with Horseradish peroxidase HRP-conjugated anti-c-Myc and total protein was visualised by Coomassie staining, (a) NbPR4 and EPO were co-expressed, EPO was tagged with MYC and detected around 35 kDa. (b) NbPR4 and Alpha-Gal were co- expressed, Alpha-Gal was tagged with MYC and detected around 55 kDa, Negative control (- ve) refers to the total extract of the leaf that was not infiltrated. Bands are annotated on the right side and position of the molecular mass marker is shown at left side (kDa), WT (Wild- type) stands for plasmid without T-DNA which was used as a control.

Erythropoietin (EPO) accumulates to higher levels when co-expressed with NbPR4 compared with the control, which was infiltrated with A. tumefaciens strain for EPO expression and untransformed A. tumefaciens. EPO was expressed with a C-terminal Myc-tag and detected with anti-c-Myc antibody around 35kDa (Example 4: NbPR4 enhances accumulation of Erythropoietin (EPO) and Alpha-galactosidase.

a) . Alpha-galactosidase (Alpha-Gal) accumulated more when co-expressed with NbPR4 compared to the control. Alpha-Gal was expressed with a C-terminal Myc-tag and detected with anti-c-Myc antibody around 55kDa (Example 4: NbPR4 enhances accumulation of Erythropoietin (EPO) and Alpha-galactosidase.

b) .

Example 5: NbPR4 enhances accumulation of VRC01, EPO and Alpha-Gal upon co- expression of all three-recombinant proteins.

To facilitate quick screening of inhibitors for stabilisation of multiple recombinant proteins, we proposed to transiently express a mixture of all three-recombinant proteins (VRC01 , EPO and Alpha-Gal along with NbPR4) in N. benthamiana. A mixture of "VRC01" HC+LC, EPO and Alpha-Gal were transiently expressed in N. benthamiana. Full leaf extract was prepared in PBS at pH=7.4 after 3dpi, total leaf extract was submitted to SDS 4-15% PAGE under reducing and non-reducing conditions. For Immunoblot analysis proteins were electro-transferred onto PVDF membrane, and total protein was visualised by Coomassie staining. Figure 5 panel (a) shows an immunoblot for representative of full size VRC01 , total leaf extract was run under non-reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti- Human Kappa to detect light chain, positive control (+ve) refers to the purified antibody and negative control (-ve) refers to total leaf extract of the non-infiltrated leaf. Figure 5 panel (b) shows an immunoblot for representative of Heavy chain, total leaf extract was run under reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-Human Gamma to detect heavy chain. Figure 5 panel (c) shows an immunoblot for a representative of EPO and Alpha-Gal, total leaf extract was run under reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-c-Myc, EPO was detected around 35 kDa, and Alpha-Gal at 55 kDa, negative (-ve) control refers to non-infiltrated leaf. Bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control. Co-expression of NbPR4 with VRC01 , EPO and Alpha-Gal simultaneously in the same plant resulted in higher yield, although yield was still lower compared to when each of the three recombinant proteins were co-expressed with NbPR4 separately (Error! Reference source not found.). These data indicate that NbPR4 enhances the accumulation of VRC01 , EPO and Alpha-Gal upon co- expression in N. benthamiana.

Example 6: C-Terminal FLAG tag disrupts NbPR4 functionality.

To investigate localization and target proteases of NbPR4, we expressed it with a C-terminal FLAG-tag. This will allow us to detect NbPR4 on immunoblots with anti-Flag antibodies. In addition, fusing NbPR4 to a FLAG-tag would allow for co-immunoprecipitation (Co-IP) of NbPR4 target proteases. Therefore, an A. tumefaciens strain for expression of NbPR4 FLAG- tag was generated. To test if NbPR4 FLAG-tag enhances recombinant protein production, VRC01 , EPO and Alpha-Gal were transiently expressed with or without NbPR4-FLAG. As control, we used plants infiltrated with A. tumefaciens strains coding the proteins of interest and untransformed A. tumefaciens.

A mixture of "VRC01" (HC+LC), EPO and Alpha-Gal were transiently expressed in N. benthamiana. Full leaf extract was prepared in PBS at pH=7.4, total leaf extract was submitted to SDS 4-15% PAGE under reducing and non-reducing conditions. For Immunoblot analysis proteins were electrotransferred onto PVDF membrane, and total protein was visualised by Coomassie staining. Figure 6 panel (a) shows an immunoblot for representative of full-size VRC01 , total leaf extract was run under non-reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-Human Kappa to detect light chain. Figure 6 panel (b) shows an immunoblot for representative of EPO and Alpha-Gal, total leaf extract was run under reducing conditions and probed with Horseradish peroxidase HRP-conjugated anti-c-Myc, EPO was detected around 35 kDa and Alpha-Gal at 55 kDa, negative (-ve) control refers to non-infiltrated leaf. Bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

Co-expression of NbPR4 FLAG-tag showed no enhanced production of VRC01 , EPO and Alpha-Gal as compared to the control (Error! Reference source not found.). This data indicates that the C-Terminal is crucial for NbPR4 functionality and appending a FLAG-tag disrupts its function.

Example 7: NbPR4 FLAG-tag and non-tag accumulate in the apoplastic fluid.

To check if NtPRI a signal peptide targets NbPR4 to the apoplast, NbPR4 FLAG-tag and NbPR4 were transiently expressed. To enhance protein expression, A. tumefaciens GV3101 encoding the silencing repressor p19 was co-infiltrated in the plants.

NbPR4 FLAG-tag and NbPR4 non-tag with p19 (RNA silencing suppressor) were transiently expressed in N. benthamiana. Apoplastic fluid was extracted in PBS at pH=7.4 after 5dpi. Apoplastic fluid was submitted to SDS 4-15% PAGE under reducing conditions. NbPR4 molecular weight corresponds to 15 kDa. Figure 7 panel (a) shows an immunoblot for the expression of NbPR4 in N. benthamiana, for Immunoblot analysis proteins, were electrotransferred onto PVDF membrane and probed with Horseradish peroxidase HRP- conjugated anti-FLAG to detect NbPR FLAG-tag and total protein was visualized by Coomassie staining, NbPR4 FLAG-tag was detected around 15 kDa. Figure 7 panel (b) shows apoplastic fluid submitted to SDS 4-15 % PAGE under reducing condition stained with SYPRO RUBY protein gel, NbPR4 FLAG-tag and NbPR4 non-tag was detected around 15 kDa. Molecular mass marker is shown on the left, WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

In the immunoblot, a 15 kDa band was detected, indicating the presence of NbPR4 FLAG-tag (Error! Reference source not found. a). Taken together, NbPR4-FLAG tag loses its functionality to stabilise recombinant proteins, but it still localises in apoplast. With this result, we cannot argue that NbPR4 non-tagged acted similarly to NbPR4 FLAG-tagged, moving into the apoplast. To address this question, the same samples were submitted to SDS PAGE 4-15% in reducing conditions, and SYPRO Ruby Protein Gel Stain was performed. A band around 15 kDa was observed in all lanes, except for the control, where plants were infiltrated with A. tumefaciens strain for p19 expression and untransformed A. tumefaciens (Error! Reference source not found. b). These 15 kDa bands correspond to NbPR4 and indicates that FLAG- tagged and non-tagged NbPR4 accumulated in the apoplast. The difference between the level of FLAG-tagged and non-tagged NbPR4 could be because the non-tagged NbPR4 functions as a protease inhibitor and is cleaved during its interaction with the protease while the non-functional FLAG-tagged NbPR4 does not get cleaved and accumulates at higher level.

Example 8: Co-expression of protease inhibitors enhances accumulation of VRC01, EPO and Alpha-Gal.

In our research group, different protease inhibitors have been tested, and have successfully shown enhanced recombinant protein production upon co-expression (unpublished). TIMP metallopeptidase inhibitor 2 is a protease inhibitor in humans. Tomato SICYS8 is a cysteine protease inhibitor that stabilises recombinant proteins in plants. MER412288 is a predicted serine protease inhibitor. The transcript encoding for MER412288 in Arabidopsis thaliana is depleted upon exposure to A. tumefaciens, as shown by a meta-analysis of four published microarray datasets (NCBI GEO numbers GSE41 16,GSE14106, GSE48402, GSE62751) (F. Grosse-Holz, unpublished). We reasoned that overexpression of N. benthamiana MER412288 might drastically shift the equilibrium of proteases and inhibitors in agroinfiltrated plants. NbPR4 FLAG-tag is used as a negative control, because of its dysfunctionality to enhance recombinant protein production (Error! Reference source not found.). All these inhibitors were cloned into the same binary plasmid used for NbPR4 expression and expressed in the plants using A. tumefaciens GV3101 strains carrying the respective plasmid for each inhibitor.

To compare the ability of NbPR4 to enhance recombinant protein production with different protease inhibitors, NbPR4, NbPR4 FLAG-tag, TIMP2, SICYS8, MER412288 and VRC01 (HC+LC) were transiently expressed.

Figure 8 panel (a) shows "VRC01" HC+LC and different protease inhibitors were transiently expressed in plants. Full leaf extract was prepared in PBS at pH=7.4 after 5dpi. Total leaf extract was submitted to SDS 4-15% PAGE under non-reducing conditions. For Immunoblot analysis proteins were electrotransferred onto PVDF) membrane and probed with Horseradish peroxidase HRP-conjugated anti-Human Kappa to detect light chain and total protein was visualised by Coomassie staining. Figure 8 panel (b) shows EPO and Alpha-Gal and different protease inhibitors were transiently expressed in plants. Total leaf extract was submitted to SDS 4-15% PAGE under reducing conditions. For Immunoblot analysis proteins were electrotransferred onto PVDF membrane and probed with Horseradish peroxidase HRP- conjugated anti-c-Myc and total protein was visualised by Coomassie staining. EPO was detected around 35 kDa and Alpha-Gal at 55 kDa. Bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

All four protease inhibitors enhanced production of VRC01 when co-expressed, except for NbPR4 FLAG-tag and the control, which was infiltrated with A. tumefaciens strains for VRC01 expression and untransformed A. tumefaciens (Figure 8a). Furthermore, co-expression of NBPR4, TIMP2 and MER412288 resulted in higher accumulation of VRC01 compared to the SICYS8. This data suggests that co-expression of all protease inhibitors resulted in higher level of VRC01 accumulation, possibly because of inhibitory activity against protease.

To investigate if the different protease inhibitors also enhance the production of EPO and Alpha-Gal, both recombinant proteins were transiently co-expressed with these inhibitors. Co- expression of NbPR4 and TIMP2 resulted in higher yield of EPO and Alpha-Gal as compared to the SICYS8 and MER412288 (Figure 8b). No accumulation of EPO and Alpha-Gal was detected when plants were co-expressed with NbPR4 FLAG-tag or the control which was infiltrated with A. tumefaciens strains for EPO and Alpha-Gal expression and untransformed A. tumefaciens. These data suggest that co-expression of protease inhibitors (NbPR4,TIMP2,SICYS8,MER412288) resulted in higher yield of EPO and Alpha-Gal as compared to the control and NbPR4 FLAG-tag. Moreover, NbPR4 and TIMP2 are superior to SICYS8 and MER412288 with regard to enhancing the accumulation of EPO and Alpha-Gal.

Example 9: NbPR4 does not affect cysteine protease activity or extracellular cysteine protease activity profile of N. benthamiana.

In the previous experiments, it has been shown that the co-expression of NbPR4 stabilises recombinant proteins. However, we still do not know the mechanism behind this phenomenon. Without wishing to be bound by any particular theory, we suspect it is due to its nature as a protease inhibitor. To investigate this, we performed an Activity-based protein profiling (ABPP). ABPP is a technique that requires a chemical probe to detect active proteomes in a living system. Different probes targeting different classes of enzymes can be used. The labelling done by ABPP is covalent and mechanism-dependent. Wth this analysis, we intended to characterise NbPR4 and find out whether it possesses the ability to inhibit serine or cysteine proteases.

CaPR4c gene in pepper is known to act as a cysteine protease inhibitor, and NbPR4 is a N. benthamiana protein with 86 % of identical amino acids compared to CaPR4. To test whether

NbPR4 also inhibits cysteine proteases, NbPR4 non-tag or NbPR4 FLAG-tag were transiently expressed in N. benthamiana. The ABPP profiling was performed using a MV201 probe, which labels papain like cysteine proteases (PLCPs). MV201 was used to monitor the activity of the cysteine proteases in total leaf extract and apoplastic fluid.

Figure 9 shows the effect of NbPR4 on the activity profile of cysteine protease of N. benthamiana. NbPR4 FLAG-tag and NbPR4 non-tag were transiently expressed in plants. Full leaf extract was prepared in sodium acetate (50mM, pH=5.0) after 5dpi and incubated for four hours with E64 (specific PLCPs inhibitor) or MV201 (PLCPs specific probe). After four hours of incubation, the reaction was stopped by adding cold acetone and submitted to SDS 4-15% and analysed by fluorescent scanning and total protein was visualised by Coomassie staining. No probe control refers to the absence of MV201 and Inhibitor refers to the addition of E64. Putative PLCPs bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

Figure 10 shows the effect of NbPR4 on extracellular cysteine protease activity profile of N. benthamiana. NbPR4 FLAG-tag and NbPR4 non-tag were transiently expressed in plants. Apoplastic fluid was extracted and prepared in sodium acetate (50mM, pH=5.0) after 5dpi and incubated for four hours with E64 (specific PLCPs inhibitor) or MV201 (PLCPs specific probe). After four hours of incubation, the reaction was stopped by adding cold acetone and submitted to SDS 4-15% and analysed by fluorescent scanning and total protein was visualised by Coomassie staining. No probe control refers to the absence of MV201 and Inhibitor refers to the addition of E64. Putative PLCPs bands are annotated on the right side and position of the molecular mass marker is shown at left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

The activity profile of cysteine protease did not change in the total leaf extract (Error! Reference source not found.) and apoplastic fluid (Error! Reference source not found.) of plants compared with the control, which was infiltrated with A. tumefaciens strain for p19 expression and untransformed A. tumefaciens. Results of ABPP suggest that NbPR4 does not affect the cysteine protease activity profile in N. benthamiana labelled with an MV201 probe. The reason we cannot see inhibition on PLCPs band could be that some cysteine proteases are not labelled with the MV201 probe, or the mode of action of NbPR4 is different from CaPR4. Also, CaPR4^'s ability to inhibit cysteine protease was detected while expressed in Escherichia Coli but not while expressed in plants via A. tumefaciens.

Example 10: NbPR4 does not affect serine protease activity or extracellular serine protease activity profile of N. benthamiana. To investigate if NbPR4 may inhibit another family of proteases, ABPP was carried using FP- RH probe, which labels active serine hydrolases. Therefore, it was used to monitor the activity of serine hydrolase in the total leaf extract and leaf apoplast of the same plants used for Example 9.

Figure 11 shows the effect of NbPR4 on serine protease activity profile of N. benthamiana. NbPR4 FLAG-tag and NbPR4 non-tag were transiently expressed in plants. Full leaf extract was prepared in sodium acetate (50mM, pH=5.0) after 5dpi and incubated for one hour with Aprotinin (Inhibitorfor serine hydrolases) or FP-Rh (Specific probe for serine hydrolases). After one hour of incubation, the reaction was stopped by adding cold acetone and submitted to SDS 4-15% and analysed by fluorescent scanning and total protein was visualised by Coomassie staining. No probe control refers to the absence of FP-Rh and Inhibitor refers to the addition of Aprotinin. Putative Serine Hydrolase bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild-type) stands for plasmid without T-DNA which was used as a control.

Figure 12 shows the effect of NbPR4 on extracellular serine protease activity profile of N. benthamiana. NbPR4 FLAG-tag and NbPR4 non-tag were transiently expressed in plants. Apoplastic fluid was extracted and prepared in sodium acetate (50mM, pH=5.0) after 5dpi and incubated for one hour with Aprotinin (Inhibitor for serine hydrolases) or FP-Rh (Specific probe for serine hydrolases). After four hours of incubation, the reaction was stopped by adding cold acetone and submitted to SDS 4-15% and analysed by fluorescent scanning and total protein was visualised by Coomassie staining. No probe control refers to the absence of MV201 and Inhibitor refers to the addition of E64. Putative Serine Hydrolase bands are annotated on the right side and position of the molecular mass marker is shown at the left side (kDa), WT (Wild- type) stands for plasmid without T-DNA which was used as a control.

There was no observed difference in serine hydrolase profiling on the total leaf extract (Figure 11) and apoplastic fluid (Figure 12) of plants compared with the control, which was infiltrated with A. tumefaciens strain for p19 expression and untransformed A. tumefaciens. This data could either signify that NbPR4 also did not affect the activity of serine hydrolase in N. benthamiana or the used probe cannot label all serine proteases.

Conclusion and Application of NbPR4

In the foregoing examples, it has been shown that NbPR4 enhances the accumulation of all three studied recombinant proteins (VRC01 , EPO and Alpha-Gal) upon co-expression in N. benthamiana. Also, NbPR4 was superior to other protease inhibitors regarding stabilising recombinant proteins upon co-expression. NbPR4 is believed to be cysteine proteases inhibitor, but we were unable to observe this with activity based protein profiling. In conclusion, NbPR4 showed great potential to enhance the yield of recombinant protein production upon co-expression, although its operating modus remains unclear.

The effects of NbPR4 are not limited to the RPs demonstrated in the examples and the protease inhibitor is broadly applicable. It can therefore be used as a tool to enhance the production of other important pharmaceutical recombinant proteins.

Example 11 : Three new protease inhibitors that can enhance accumulation of recombinant proteins in molecular farming.

a. Finding plant inhibitors that are depleted upon interaction with A. tumefaciens

We began the search for protease inhibitors by screening the literature for putative candidates and testing whether their co-expression boosts RP accumulation. The initial aim was to identify candidate inhibitors for co-expression in molecular farming among the 11 1 predicted protease inhibitors in N. benthamiana, reasoning that endogenous sequences would be less problematic to express than foreign ones. It was hypothesised that maximum impact on the proteolytic activity in the apoplast from inhibitors which would not be present or very low abundant under natural conditions. Attention was focused on inhibitors depleted upon agroinfiltration. Inhibitors depleted in different plant species upon interaction with A. tumefaciens, were identified, following the reasoning that any positive effects on RP accumulation would be readily transferrable between plant expression platforms (different species) by using the respective orthologs of the inhibitors. To identify depleted inhibitors, a meta-analysis of public datasets on the plant response to A. tumefaciens was performed. Datasets deposited in the NCBI GEO database were filtered for the organism group "green plants" (taxid 33090) and the term "Agrobacterium" (543 hits). The first 53 hits were full studies, the rest referred to the single replicates from these studies. There were no studies on N. benthamiana. Four microarray studies of A. thaliana infected with A. tumefaciens were chosen for further analysis:

GSE4116: Analysis of A. thaliana suspension cells up to 48 hours after infection with oncogenic Agrobacterium tumefaciens strain A348

GSE14106: A. thaliana plants infected with GV3101 (disarmed A. tumefaciens strain)

GSE48402: A. thaliana plants wounded and infected with A. tumefaciens C58

GSE62751 : A. thaliana seedlings in hydroponic culture; A. tumefaciens C58 was inoculated into the hydroponic solution Data from the selected studies were analysed in GE02R, comparing the treated vs non- treated replicates in two groups. Thus, timecourses were averaged where they had been part of the experimental design. The "top 250" analysis was carried out for each dataset using the standard settings. The resulting genes were filtered for those containing the term "inhibit*" in their gene description. The protein sequences for the resulting genes were retrieved from the TAIR database. A BLASTP search was performed to search for orthologs of these A. thaliana protease inhibitors in N. benthamiana, using the N. benthamiana protease inhibitor sequences deposited in the MEROPS database (www.ebi.ac.uk/merops). For dataset GSE48402, no ATG numbers were given in the spot descriptions. Thus, a TBLASTN search was performed, using the N. benthamiana inhibitor protein sequences and the nucleotide sequences given for the top 250 spots. To account for the short nucleotide sequences (most of them 70 residues), BLAST parameters were adjusted to PAM30 Matrix, gap cost existence: 10, extension: 1. The best N. benthamiana BLAST hits of each spot sequence were considered orthologous to the transcripts monitored on that spot. Where unique orthologous N. benthamiana inhibitors from the MEROPS set and their corresponding genes in the Niben101 genome (www.solgenomics.com) could be identified, the logFC values for the A. thaliana transcripts were obtained from the microarray data and the profiles examined visually to select transcripts depleted upon interaction with A. tumefaciens. This resulted in 10 candidate inhibitors (4 x I3, 3 x 113, 2 x I25, 1 x I20), of which five were selected to represent each inhibitor family at least once: MER062480 (I20; the same protein had been selected previously as it is very similar to a N. alata protein that enhances recombinant protein accumulation in rice suspension cells upon co-expression (Kim et ai, 2008); named NbPot2), MER41 1712 (I25), MER41 1950 (I3), MER412033 (113), MER41 1832 (113) and MER412288 (113, later named NbPotl). Three 113 inhibitors were chosen because they shared less than 50 % identical amino acids with each other.

b. Co-expression of NbPR4, NbPotl or HsTIMP2 enhances accumulation of aGal, EPO and VRC01

29 putative inhibitors were selected, cloned and tested, 26 of which did not boost RP accumulation, even when directed to the apoplast by fusion to the NtPR1 signal peptide. Three of the tested putative protease inhibitors did boost RP accumulation when fused to the signal peptide (SP) of the NtPR1 gene (Figure 21).

Figure 21 shows co-expression of NbPR4, NbPotl or HsTIMP enhances accumulation of aGal (a), EPO (b) and VRC01 (c). Leaves were infiltrated with 1/1 (v/v) mixes of A. tumefaciens strains carrying plasmids for EPO (a) or aGal (b) and inhibitor expression or 1/1/1 (v/v) mixes of A. tumefaciens strains carrying plasmids for VRC01 heavy chain, VRC01 light chain and inhibitor expression (c). Full leaf extracts were harvested at 3 dpi. Proteins were subjected to reducing (a-b) or non-reducing (c) SDS-PAGE and transferred onto PVDF membranes. EPO (a), aGal (b) and VRC01 (c) accumulation was ualized using the indicated antibodies, all of which were fused to horseradish peroxidase (HRP). Closed and open triangles in (c) indicate the full length VRC01 and degradation products, respectively. Intensities of the double band in (a), the band in (b) and the top band in (c) were quantified using ImageJ and normalized to the highest intensity within each blot. The blots are representative for at least six biological replicates each.

This work reveals three new, unrelated protease inhibitors that can enhance accumulation of RP in molecular farming. All three inhibitors were tested in recombinant protein accumulation assays multiple times. They reproducibly enhance the accumulation of VRC01 , EPO and aGal and reproducibly show additive effects (shown in at least 3 independent experiments).

In the case of HsTIMP, a mutant version of the protein Ala-HsTIMP) that has been characterized as lacking protease inhibitor function in animal cells (Wingfield et al., 1999) was tested for its effect on RP expression. The inactive Ala-HsTIMP does not boost recombinant protein accumulation upon co-expression, indicating that metalloprotease inhibitor function is required for this effect (Figure 21).

The invention to boost RP accumulation in N. benthamiana by co-expression of these three NtPRISP-inhibitor fusion proteins, which were studied further and are described in more detail below.

NbPR4 is a homolog of CaPR4, which was recently shown to be a protease inhibitor (Kim & Hwang, 2015). When expressed with its native signal peptide, NbPR4 co-expression does not boost RP accumulation. However, when its signal peptide (SP) is replaced with that of the NtPR1 gene, NbPR4 co-expression does boost RP accumulation, which was an unexpected discovery. Advantageously, the NtPR1 SP-NbPR4 fusion can now be applied to boost recombinant protein accumulation upon transient co-expression with RP.

A protein sequence analysis was conducted and indicated that NbPotl may function as a serine and cysteine protease inhibitor (MEROPS family 113) from N. benthamiana. A metaanalysis of public transcript-level data, deposited in NCBI under GEO numbers

GSE41 16, GSE14106, GSE48402, GSE62751 revealed that a transcript that is highly similar to the one encoding NbPotl is depleted in Arabidopsis upon exposure to Agrobacterium tumefaciens. The publically available data does not indicate whether NbPotl acts as a protease inhibitor and does not link it to transient protein expression. NbPotl does not have a predicted signal peptide for secretion, so a NtPR1 SP-NbPot1 fusion protein was generated to change the subcellular localization of NbPotl Furthermore, two other putative 113 protease inhibitors and five more putative Ser and Cys protease inhibitors (MEROPS family I3) were tested by generating NtPRI SP- fusions; none of these boosted RP accumulation. Advantageously, the NtPR1SP-NbPot1 fusion can now be applied to boost recombinant protein accumulation upon transient co-expression with RP.

HsTIMP2 is a human metalloprotease inhibitor that is being investigated as a potential anticancer agent (Wingfield et al., 1999; Arkadash et al., 2017). HsTIMP2 has never been expressed in plants. To test whether it could be useful in increasing the RP accumulation in plants, HsTIMP2 was fused to the NtPR1 signal peptide. It was found that the NtPRI SP- HsTIMP2 fusion boosts RP accumulation on co-expression. The application of a human metalloprotease inhibitor to boost recombinant protein accumulation levels upon transient co- expression in N. benthamiana is surprising given that even members of the same class of endogenous plant protease inhibitors (e.g. the 113 inhibitors) are not all capable of increasing RP levels in plants.

A > 10-fold recombinant protein level upon expression of each inhibitor separately was detected (Figure 21). The tested recombinant proteins are: alpha-galactosidase (against Fabry disease), erythropoietin (EPO, against anemia), and VRC01 antibody (both heavy and light chains, HC/LC, against HIV). The strategy of co-expressing protease inhibitors to boost RP accumulation is known and has been successfully applied by others (Goulet et al., 2012; Jutras et al., 2016), but NbPR4 and HsTIMP are superior in boosting RP accumulation when compared to known protease inhibitor SICYS8 (Jutras et al., 2016).

Example 12: Combinations of NbPR4, HsTIMP and SICYS8 further enhance accumulation of aGal, EPO and VRC01

An additive effect was observed when these inhibitors were combined (Figure 22). Figure 22 shows combinations of NbPR4, HsTIMP and SICYS8 enhance accumulation of aGal, EPO and VRC01. Leaves were infiltrated with 1/1 (v/v) mixes of A. tumefaciens strains carrying plasmids for EPO (a) or aGal (b) and PI expression or 1/1/1 (v/v) mixes of A. tumefaciens strains carrying plasmids for VRC01 heavy chain, VRC01 light chain and PI expression (c). The PI part of the mixture contained three volumes of A. tumefaciens strains for expression of the indicated Pis, with one part Ala-HsTIMP used in lanes 2-4, two parts Ala-HsTIMP in lanes 5-7 and three parts Ala-HsTIMP in lane 8 to replace the missing Pis. Full leaf extracts were harvested at 3 dpi. Proteins were subjected to reducing (a-b) or non-reducing (c) SDS-PAGE and transferred onto PVDF membranes. EPO (a), aGal (b) and VRC01 (c) accumulation was visualized using the indicated antibodies. Intensities of the double band in (a), the band in (b) and the top band in (c) were quantified using ImageJ and normalized to the highest intensity within each blot. The blots are representative for at least six biological replicates each.

Example 13: Intact and mutant Inhibitor proteins accumulate in N. benthamiana leaves upon transient expression

Figure 23 shows intact and mutant inhibitor proteins accumulate in N. benthamiana leaves upon transient expression. Inhibitor-derived peptides (blue) detected by MS in agroinfiltrated leaves expressing p19 and each of the inhibitors, mapped to the inhibitor sequences (black) which are fused to the NtPR1 signal peptide (red). For HsTIMP and SICYS8, peptides that are specific to the intact or mutant inhibitor, respectively, are marked in light blue and mutations are indicated by green dots.

Example 14: Asp and Ser proteases are depleted upon NbPR4 and NbPotl overexpression, while proteins associated with defence signalling accumulate upon NbPR4, NbPotl , HsTIMP and SICYS8 overexpression

Figure 24 shows Asp and Ser proteases are depleted upon NbPR4 and NbPotl overexpression, while proteins associated with defence signalling accumulate upon NbPR4, NbPotl , HsTIMP and SICYS8 overexpression. Label-free quantitative MS shows proteins that differ significantly (t-test, p<0.05) and more than two-fold in abundance between inhibitor overexpressing and control (p19 expressing) leaves. Representative proteins of interest are annotated in colour; proteins that change less than two-fold and/or non-significantly are not shown.

Example 15: In vitro proteolytic degradation of VRC01 is inhibited by CYS8

Figure 25 shows in vitro proteolytic degradation of VRC01 is inhibited by CYS8. Plant- produced VRC01 fluorescently labelled on lysine residues (flou-VRC01) was spiked into leaf extracts (200 ng/mL) and incubated at 37 C. Leaf extracts were pre-incubated with either a chemical inhibitor cocktail or solvent mix. Flou-VRC01 was visualized by SDS-PAGE and in- gel fluorescence scanning (a-b). Leaves were infiltrated with A. tumefaciens harbouring the p19 expression plasmid (a), mixed 1/1 (v/v) with A. tumefaciens harbouring the respective protease inhibitor (PI) expression plasmid (b). The mixed sample in (b) contained equal volumes of all inhibitor-expressing samples and the p19 control, but no inhibitor cocktail. Fluorescence intensity of the VRC01 heavy chain (HC) band was quantified using ImageJ (c). Intensities were normalized to the intensity at 0 h to obtain recovery values, bars represent the average and error bars the standard deviation across three biological replicates (independent agroinfiltrations) (c). * = significantly different from p19 control, t-test p<0.05. Closed triangles indicate VRC01 heavy and light chains (HC and LC), open triangles mark putative degradation products.

Example 16: Activity-based protein profiling reveals that NbPR4, NbPotl and HsTIMP differ from SICYS8 in their mode of action

Figure 26 shows activity-based protein profiling reveals that NbPR4, NbPotl and HsTIMP differ from SICYS8 in their mode of action. Activity-based profiling of papain-like Cys proteases (a), Ser hydrolases (b) and vacuolar processing enzymes (c). Leaves were infiltrated with A. tumefaciens harbouring the indicated protease inhibitor (PI) expression plasmid, mixed 1/1 (v/v) with A. tumefaciens harbouring the p19 expression plasmid. Leaf extracts (pH 5) were obtained at 4 dpi, adjusted to the same protein concentration and 48 μΙ of each sample were incubated with or without the respective probe for 4 h (a-b) or 1 h (c) at room temperature. The reaction was terminated by acetone precipitation, proteins were subjected to reducing SDS-PAGE and active enzymes visualized by in-gel fluorescence scanning.

Example 17: PI must be expressed in the apoplast to enhance secretory RP accumulation in plants.

Figure 27 shows that fusion of NtPR1 signal peptide to NbPR4 enhances VRC01 accumulation.

Example 18: Degradation of both fluorescent and unlabelled VRC01 happens in vitro and is limited by the inhibitor cocktail.

Figure 28 shows degradation of both fluorescent and unlabelled VRC01 happens in vitro and is limited by the inhibitor cocktail.

Example 19: General Materials and Methods

All chemicals were obtained from Sigma (Sigma-Aldrich, St. Louis, US) unless specified otherwise. a. Cloning of inhibitor expression plasmids

The MoCIo plant parts kit (Engler ef o/., 2014) was used for cloning and all vectors are from this kit unless specified otherwise. The sequence encoding NbPR4 was amplified from N. benthamiana genomic DNA using primers #001 (including the native SP) or #005 (without the native SP) and #003 and cloned into pLOV-SC-41308 (including the native SP) or pLOV-C- 41264 (without the native SP), respectively. The sequence encoding HsTIMP was codon- optimized for /^'n planta expression, synthesized as GeneStrings (Thermo Fisher Inc, Waltham, US) and cloned into pLOV-SC-41308 (including the native SP) or pLOV-C-41264 (without the native SP), respectively. The sequence encoding NbPotl was amplified from N. benthamiana genomic DNA using primers #074 and #075 and cloned into pLOV-C-41264. Each inhibitor level 0 module was then combined with pL0M-PU-35S-TMV-3-51288, pL0M-S-NtPR1a SP (X06361) PIV2 (pJK002, which was a gift from Jiorgos Kourelis (Addgene plasmid # xxx), PR1 signal peptide from Nicotiana tabaccum), pL0M-T-35S-1-41414 and pL1VB-F-pAGM4723 in a Bsal reaction to obtain the expression plasmids pFGH008 (NbPR4), pFGH053 (NbPotl) and pFGH047 (HsTIMP). Plasmids were transformed to E. coli for amplification, purified, sequenced and transformed to Agrobacterium GV3101-pMP90. Agrobacterium GV3101- pMP90 were cultured on plates of LB medium (10 g/L NaCI, 10 g/L Tryptone, 5 g/L yeast extract, 15 g/L agar) containing 25 μΜ Rifampicin, 50 μΜ Gentamycin and 50 μΜ Kanamycin to select for transformants. A single colony was picked and cultured in liquid LB medium (10 g/L NaCI, 10 g/L Tryptone, 5 g/L yeast extract) containing 25 μΜ Rifampicin, 50 μΜ Gentamycin and 50 μΜ Kanamycin. Glycerol stocks were prepared by mixing this culture 1/1 (v/v) with 50 % glycerol in water, flash-freezing in liquid Nitrogen and storing at -80 °C. For each agroinfiltration experiment, a 160 μί aliquot of glycerol stock was thawed and inoculated into fresh LB.

b. Agroinfiltration procedure

N. benthamiana plants were grown at 21 °C under a 16/8 h light/dark regime in a growth room. Agrobacterium GV3101-pMP90 (WT), Agrobacterium GV3101-pMP90 carrying a Pig- encoding plasmid (pJK050, which was a gift from Jiorgos Kourelis (Addgene plasmid # 101751), P19 from tomato bushy stunt virus) orAgro 5acter/i m GV3101-pMP90 carrying EPO, aGal, VRC01 or inhibitor encoding plasmids were grown for 21 h at 28 °C with agitation in LB containing 25 μΜ Rifampicin and 50 μΜ Gentamycin (for WT) plus 50 μΜ Kanamycin (for A. tumefaciens harbouring plasmids). Bacteria were collected by centrifugation at 2000 g for 5 min at room temperature (RT), resuspended in infiltration buffer (10 mM 2-(N- morpholino)ethanesulfone (MES), 10 mM MgC , pH 5.7, 100 μΜ acetosyringone) to Οϋβοο = 0.5 (OD6oo = 1 for aGal) and left for 2 h at 28 °C with agitation to recover. For co-expression experiments, A. tumefaciens suspensions were mixed in the appropriate ratios (described in figure legends). The first and second fully expanded leaves of pre-flowering stage N. benthamiana (4-5 weeks old) were infiltrated with the bacteria suspension using a syringe without a needle. For comparison, different A. tumefaciens suspension mixes were infiltrated into different sectors of the same leaf. c. Protein extraction and detection by Western Blot

Leaf extracts were prepared at three days post infiltration (dpi). Four leaf discs (22 mg each) from four individual plants were combined per sample, flash-frozen in liquid Nitrogen and pulverized in a TissueLyser ball mill (Qiagen, Hilden, DE). The tissue powder was mixed with 3/1 (v/fresh weight) cold PBS and centrifuged for 10 min at 16.000 g and 4 °C. The supernatant was mixed with 4x gel loading buffer (200 mM Tris-HCI (pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol), heated for 5 min at 95 °C and separated on Bis-Tris gels at 100 V. Proteins were then transferred to a PVDF membrane using the TransBlot Turbo system (Biorad, Hercules, US). The membrane was blocked in 5 % milk in TBS (50 mM Tris-CI, pH 7.6; 150 mM NaCI) for 1 h at room temperature (RT), incubated in 1/5000 anti-myc-HRP (ab1326, Abeam, Cambridge, UK) for detection of aGal and EPO or in 1/2000 anti-kappa-HRP (Sigma A7164) or 1/2000 anti-gamma-HRP (Sigma A6029) for detection of VRC01 over night at 4 °C and washed in TBST (0.005 % Tween-20) prior to detection with Clarity ECL substrate (BioRad). d. Protein extraction and ABPP/spike-in assays

Leaf extracts were prepared at four days post infiltration (dpi), to allow for maximum protein accumulation in the presence of the p19 silencing suppressor. Four leaf discs (22 mg each) from four individual plants were combined per sample, flash-frozen in liquid Nitrogen and pulverized in a TissueLyser ball mill (Qiagen, Hilden, DE). The tissue powder was mixed with 3/1 (v/fresh weight) cold 50 mM NaAc, pH 5, 500 mM DTT and centrifuged for 10 min at 16.000 g and 4 °C. The supernatant was used for ABPP and spike-in experiments as described in the figure legends. Reactions were terminated by adding 1 mL cold actetone. Samples were centrifuged for 3 min at 16000 g, the supernatant discarded and the proteins resuspended in 2 x gel loading buffer, heated for 5 min at 95 °C and separated on Bis-Tris gels at 100 V. Flourescence scanning was performed on a Typhoon scanner (Amersham/GE Healthcare, Little Chalfont, UK). e. Primers

Table 1. Primers used for NbPR4 amplification

Name Sequence #001#NbP 4_natSP_FW GATCGATCTTGAAGACAAAATGGAGAGAGTAAATAATTACTATAAG (SEQ ID NO: 36)

#003#NbPR4_REV TTGAAGACAAAAGCTTAGTCATCGCAGTTGATAAATTCATAG

(SEQ ID NO: 37)

#005#NbPR4_noSP_FW CAGAGCGCTACAAACGTGAG

(SEQ ID NO: 38)

#074#NbPotl_REV TTGAAGACAAAAGCTTAACCCACTGTGGGAGTAATTATAACG

(SEQ ID NO: 39)

#075#NbPotl_FW TTG AAG AC A A AG GTA AG C ATTTATG G CCTG AACTTGTG G (SEQ ID NO: 40)

f. Plant Material

Nicotiana benthamiana was grown in a growth chamber, with a day period consisting of 16 hours at 21 °C and an 8-hour night period at 20°C. Plants with 4-5 weeks and fully expanded leaves were used in the experiments. g. Apoplastic fluid extraction

Whole leaf of N. benthamiana were submerged in ice-cold water and subjected to the vacuum infiltration. To remove the air from the leave apoplastic space, vacuum was applied for ten minutes. When vacuum was released, water was allowed to enter back in. The leaves were then blotted dried and introduced in a syringe inside a centrifuge tube. Collection of apoplastic fluid was done by centrifugation at 2,000 rpm with a slow acceleration and deceleration of the rotor for 25 minutes at 4°C. h. Protein extraction

Leaf discs of 50-100 mg were sampled and flash frozen in liquid nitrogen before homogenization with metal beads and TissueLyser (Qiagen). To calculate the appropriate volume, in μΙ, of phosphate-buffered saline solution (10 mM Na₂HP04; 2.7 mM KCI; 137 mM NaCI; 1.8 mM KH2PO4) to add to each sample of tissue powder, we multiplied the fresh weight (g) of each sample by three. After the addition of phosphate-buffered saline, each sample was centrifuged (10 minutes, 4°C, 13,000 rpm), supernatants were shifted to new labelled 1.5 ml Eppendorf tubes. Reducing condition samples were prepared by adding an appropriate volume of 4X reducing gel loading buffer (400 mM DTT; 200 mM Tris-CI; 8% SDS; 0.4% 40% glycerol bromophenol blue; pH 6.8). Non-reducing conditions were prepared by addition an appropriate volume of 5X non-17 reducing gel loading buffer (250 mM Tris-CI, 10% SDS;

0.5%; 50% glycerol, bromophenol blue pH 6.8). Samples were heated up to 95°C for 5 minutes and spun down at 13,000 rpm for 5 seconds.

1. Immunoblotting

Total leaf extract/ apoplastic fluid were separated on 4-15 % SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane by electrophoresis. To block unspecific binding of monoclonal antibodies, the PVDF membrane was incubated for one hour at room temperature or 4 °C overnight with 5% milk TBS (50 mM Tris-CI, 150 mM NaCI; pH 7.5). The antibodies were prepared in 5% milk-TBS. Full length VRC01 antibody and its degradation products were detected under non-reducing conditions with horseradish peroxidase- conjugated (HRP) anti-Human Kappa. Visualization of the heavy chain was made under reducing condition with HRP-conjugated anti-Gamma. EPO and Alpha-Gal were detected under reducing conditions with HRP-conjugated anti-c-Myc. NbPR4 FLAG-tag was probed with anti-Flag under reducing conditions. j. Activity based protein profiling

For serine hydrolase activity, apoplastic fluid/ total leaf extract samples were labeled with 0.2 μΙ TAMRA-FP serine hydrolase probe (Thermo Scientific) in the presence of 5mM sodium acetate pH 5, 5mM DTT at the room temperature for one hour. For papin like cystine proteases activity, apoplastic fluid/ total leaf extract samples were labeled with 0.1 μΙ MV201 (Thermo Scientific) in the presence of 5mM DTT, 5mM sodium acetate pH 5 at the room temperature for four hours. Protein in the mixture was precipitated with ice-cold acetone and centrifuged at 13,000 rpm for 5 minutes, to stop the reaction. Resuspension of the pellet was done in 2X gel loading buffer (100 mM Tris-CI, 200 mM DTT,20% glycerol, 4% SDS, 0.2% bromophenol blue (pH 6.8)) and heated up to 95°C for 5 minutes. Samples were loaded to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) 4-15% and labeled enzymes were observed for fluorescence using Typhoon scanner (Cy3 filter, green 532 nm laser). k. Trypan blue staining

For cell death diagnosis, leaves of N. benthamiana were harvested at different time points and incubated for 5 minutes at 95°C in Trypan Blue Solution (TB). Samples were incubated overnight at room temperature. Staining solution was replaced with chloral hydrate (de- staining solution) and incubated overnight at RT. After de-staining overnight, samples were transferred to new tube containing 50% glycerol and scanned using scanner. Example 20: Sequence Requirements

NtPR1 Signal Peptide Notes:

• An intron was inserted after the signalP sequence when (and only when) NtPR1 was used to ensure translation by the plant (and not the bacteria, which cannot splice the intron and would thus encounter a double stop codon after the signal peptide)

• Whenever the NtPRISP was used, it is followed by a Gly-Gly linker - this is a feature of golden gate cloning.

• Two mutant versions were tested that do not enhance RP accumulation, replacing the N-terminus of the protein (native: CSCSP [SEQ ID NO: 3]) with ACSCSP (SEQ ID NO: 41) or ASASP (SEQ ID NO: 42), respectively. Thus, the intact N-terminus is important for function.

NbPR4 Notes:

• Appending a flag-HA tag (sequence GDYKDDDDKYPYDVPDYA (SEQ ID NO: 43), coding sequence GGTGACTACAAGGACGACGATGACAAGTACCCATACGATGTTCCAGATTACGCT TAA (SEQ ID NO: 44)) at the C-terminus or a 6xHis tag (sequence HHHHHHGG (SEQ ID NO: 45), coding sequence CATCACCATCACCATCACGGAGGT (SEQ ID NO: 46)) at the N-terminus disrupts the functionality of NbPotl in enhancing RP accumulation.

NbPotl Notes:

• Appending a flag-HA tag (sequence GDYKDDDDKYPYDVPDYA, coding sequence GGTGACTACAAGGACGACGATGACAAGTACCCATACGATGTTCCAGATTACGCT TAA) at the C-terminus or a 6xHis tag (sequence HHHHHHGG, coding sequence CATCACCATCACCATCACGGAGGT) at the N-terminus disrupts the functionality of NbPotl in enhancing RP accumulation.

HsTIMP2 Notes:

• When the two N-terminal Cys residues are mutated to Ala or Gly or an Ala is appended at the N-terminus, the functionality of HsTIMP in enhancing RP accumulation is lost.

• Appending a flag-HA tag (sequence GDYKDDDDKYPYDVPDYA, coding sequence GGTGACTACAAGGACGACGATGACAAGTACCCATACGATGTTCCAGATTACGCT TAA) at the C-terminus or a 6xHis tag (sequence HHHHHHGG, coding sequence CATCACCATCACCATCACGGAGGT) at the C-terminus disrupts the functionality of HsTIMP in enhancing RP accumulation. Example 21 : Amino Acid and Nucleotide Sequence Alignments

HsTIMP2: Figure 27 shows an alignment of HsTIMP2 (protein) with homologues from the NCBI landmark database. All the proteins included in the alignment are annotated as TIMP. The arrow markers indicate metalloprotease binding sites in the respective organisms. The lowermost three sequences are translations from the constructs used for in planta expression. The two N-terminal Cys residues (beginning of the Metztincin binding interface) are important for enhancing RP accumulation by co-expression in plants. There were no BLAST hits in the NCBI nr database when I restricted the organism group to "green plants (taxid:33090)", so it is thought that TIMPs are not conserved in plants. Figure 29 shows a matrix of the percentage of identical amino acids as between HsTIMP2 (protein) with homologues from the NCBI landmark database. Figure 31 shows an alignment of HsTIMP2 (nucleotide) with homologues from the NCBI nr database. The two N-terminal Cys residues (beginning of the Metztincin binding interface) are not conserved in the Alligator and Camel sequences. Figure 31 shows a matrix of the percentage of identical nucleotide base pairs as between HsTIMP2 (nucleotide) with homologues from the NCBI nr database.

NbPR4: Figure 33 shows an alignment of NbPR4 (protein) with homologues from the NCBI landmark and nr databases. The sequence "NtPR1aSP-NbPR4" is the translation from the construct used for in planta expression. When green plants are excluded (txid33090) from the database, the search returned no hits except the Streptomyces protein (WP_078651053), so this protein seems not to be conserved outside green plants. It may be interesting to test whether WP_078651053 can enhance RP accumulation upon co-expression in N. benthmamiana, as it lacks the N-terminus. Wthout wishing to be bound by any particular theory, based on the experiments described herein that show N- and C- terminal tags disrupt NbPR4 capacity to enhance RP accumulation upon co-expression in N. benthmamiana, it is expected that both termini are crucial and WP_078651053 could be used to test that. In the alignment, green bits in the bar above the alignment indicate identical amino acids, so the part marked in red (and other areas that are dark) seems conserved, although I cannot say what its function might be. The Chitin binding domain is not needed to enhance RP accumulation upon co-expression in N. benthmamiana. Figure 32 shows a matrix of the percentage of identical amino acids as between NbPR4 (protein) with homologues from the NCBI landmark database. Figure 33 shows an alignment of NbPR4 cDNA with homologues from the NCBI nr database. NbPR4 cDNA was blasted (without the intron). The same hits were returned for NbPR4 genomic DNA (with the intron) or with the native signal peptide. The gene seems not to be conserved outside eudicots and maybe not outside the Solanaceae, as the only hit outside that clade (AY725195, which is from a species that belongs to the Fabaceae) lacks the N-terminus. Without wishing to be bound by any particular theory the experiments described herein indicate that the N-terminus is important, because an N-terminal His-tag disrupts NbPR4 capacity to enhance RP accumulation upon co-expression in N. benthmamiana. However, this is contradictory to the sequence identity data, which show there is a 66% identical protein in Medicago (Fabaceae). A matrix of the percentage of identical nucleotide base pairs as between NrPR4 cDNA with homologues from the NCBI nr database is shown in Figure 34. This implies the protein may be conserved outside the Solanaceae, while the gene structure is not. NbPR4-like genes in other species may have other introns, which stop BLAST from recognizing them as similar.

NbPotl : Figure 46 shows an alignment of NbPotl (protein) with homologues from the NCBI landmark database. NbPotl was cloned based on MER412288, a N. benthamiana protein recorded in MEROPS. The sequence "NtPR1aSP-NbPot1" is the translation from the construct I used for in planta expression. In a N. benthamiana proteome database compiled and curated by the inventors (Grosse-Holz et al, PBJ, under review), there are two slightly longer proteins that carry the sequence of MER412288 = NbPotl , but with longer N-termini (Niben101 Scf00750XLOC_013210 and Niben101 Scf00750XLOC_013207). Neither of these has a signal peptide predicted by SignalP. The N-terminus appears conserved across a wide range of species, not only plants. A matrix of the percentage of identical amino acids as between NbPotl (protein) with homologues from the NCBI landmark database is shown in Figure 35. An alignment of NbPotl (nucleotide) with homologues from the NCBI nr database is shown in Figure 479. The N-terminus appears conserved on the DNA level across a wide range of Eudicots, including the Fabaceae, Asteraceae and Solanaceae. Figure 36 shows a matrix of the percentage of identical nucleotide base pairs as between NbPotl (nucleotide) with homologues from the NCBI nr database.

The other two 113 inhibitors in N. benthamiana (MER411832; MER412033) were not taken forward in the development of the toolbox; each of them was tested twice and neither showed a clear increase in RP accumulation upon co-expression. In order to identify sequence regions which may be important for generating increases in RP accumulation, the amino acid sequence of NbPotl was aligned with that of the other two 113 inhibitors. This alignment is shown in Figure 37. It was found that the N-terminal conserved region is shared between the

3 in, so if they don't increase RP accumulation upon co-expression, more than the conserved

N-terminal region is required. Figure 38 shows a matrix of the percentage of identical amino acids as between NbPotl (protein) with and the two other N. benthamiana 113 inhibitors. In order to identify sequence regions which may be important for generating increases in RP accumulation, the nucleotide sequence of NbPotl was aligned with that of the other two N. benthamiana 113 inhibitors. This alignment is shown in Figure 49. Figure 39 shows a matrix of the percentage of identical nucleotides as between NbPotI (nucleotide) and the two other

N. benthamiana 113 inhibitors.

In order to identify any amino acid sequence regions common to proteases which can enhance

RP in N. bentamiana, the amino acid sequences of all three protease inhibitors (HsTIMP2,

NbPR4, NbPotI) and SICYCS8 were aligned (shown in Figure 40). A matrix of the percentage of identical amino acids as between all three protease inhibitors (HsTIMP2, NbPR4, NbPotI) and SICYCS8 is shown in Figure 41.

A summary of all candidate proteins, selected, cloned and tested is provided in Table 1 below:

TABLE 1 o o 4-» ro

n.

la n. 1 E ro

Q. ro o

M o r

o in £ o

a. +

predicted A. tumefaciens PI; we reasoned overexpression might aid bacterial infection and boost pLlB-F-

AtMac 139 NtPRISP 35S pFGH057 NO transformation

AtMacroglobulin

with the RP encoding DNA. Additionally, macroglobulins are exceptionally versatile Pis targeting all classes of proteases, so we hypothesized this might inhibit any protease classes that we had not targeted directly.

predicted A. tumefaciens PI; we reasoned overexpression might aid

SI

AtSerPI pLIB-F-AtSerPI 151 NtPRISP 35S none pFGH056 NO bacterial

(?)

infection and boost transformation with the RP encoding DNA

Shindo mutant version et al. of CIPl (control)

2016.

CI, C- pLlVB-F-Cl- PLoS

CIPl 142 SI, NtPRISP 35S terminal pFGH105 NO

Cipl_deltaTT Pathog.

Al flag-HA

2016

12(9):el

005874.

Shindo

et al.

2016.

CI, C- pLlVB-F-Cl- PLoS mutant version

CI Pl 142 SI, NtPRISP 35S terminal pFGH106 NO

Cipl TTtoAA Pathog. of CIPl (control)

Al flag-HA

2016

12(9):el

005874.

pL2M-Fl-pNos_S- CI, Shindo plant pathogen-

CIPl NtPRl_C-CIPl_T- 142 SI, NtPRISP Nos none pJK138 NO et al. derived PI that

Nos Al 2016. targets plant PLoS extracellular

Pathog. proteases.

2016 Under a weaker

12(9):el promoter to

005874. avoid potential deleterious effects

Shindo

et al.

plant pathogen-

2016.

pL2M-Fl-p35S_S- CI, derived PI that

PLoS

CIPl NtPRl_C-CIPl_T- 142 SI, NtPRISP 35S none pJK139 NO targets plant

Pathog.

35S Al extracellular

2016

proteases.

12(9):el

005874.

Shindo plant pathogen- et al. derived PI that targets plant

2016.

C- extracellular

CI, PLoS

pLlVB-F-Cl- termin NO proteases.

CI Pl 142 SI, NtPRISP 35S pFG H104 Pathog.

Cipl al flag- (?)

Al 2016

HA

12(9):e

100587

4.

Tian et

al. 2004. plant pathogen-

J. Biol. derived PI that

Ser/

EPI1 pLlB-F-EPIl 11 NtPRISP 35S none pFGH048 NO Chem. targets plant

Cys

279: extracellular

26370- proteases.

26377.

plant pathogen- derived PI that

Ser/ (related

EPI12 PL1B-F-EPI12 11 NtPRISP 35S none pFGH049 NO targets plant

Cys to EPI1)

extracellular proteases.

Tian et plant pathogen- pL2M-Fl-PU-35S- NO

EPIC1 125 Cys NtPRISP 35S none pJK137 al. 2007. derived PI that

TMV_S- (?)

Plant targets plant NtPRlaPIV2_C- Physiol. extracellular EPIC1 _T-35S 143: proteases.

364- 377.

plant pathogen- derived PI that

Tian et

targets plant al. 2007.

pL2M-Fl-PU- extracellular

Plant

pNos_S- proteases;

EPIC1 125 Cys NtPRISP Nos none pJK136 NO Physiol.

NtPRlaPIV2_C- under a weaker

143:

EPIC1 _T-Nos promoter to

364- avoid

377.

deleterious effects plant pathogen- derived PI that

Tian et al. targets plant pL2M-Fl-PU-

2007. extracellular pNos_S-

Plant proteases;

EPIC2B NtPRlaPIV2_ 125 Cys NtPRISP Nos none pJK042 NO

Physiol. under a weaker

C-Epic2B_T-

143: 364- promoter to

Nos

377. avoid

deleterious effects

Tian et al.

pL2M-Fl-PU- plant pathogen-

2007.

35S-TMV_S- derived PI that

Plant

EPIC2B NtPRlaPIV2_ 125 Cys NtPRISP 35S none pJK148 NO targets plant

Physiol.

C-EPIC2B _T- extracellular

143: 364- 35S proteases

377.

pL2M-Fl-PU- Lozano- plant pathogen-

35S-TMV_S- Torres et

derived PI that

NtPRlaPIV2_ al. 2012.

GrVAPl ? Cys NtPRISP 35S none pJK141 NO targets plant

C-Gr-VAP-1 PNAS 109:

extracellular

(E.coli)_T- 10119- proteases

35S 10124.

plant pathogen-

PL2M-F1- Lozano-

NO d PI that

GrVAPl ? derive

PU-pNos_S- cys NtPRISP Nos none pJK140 Torres et

(?) targets plant

NtPRlaPIV2 al. 2012.

extracellular _C-Gr-VAP-1 PNAS proteases;

(E.coli) T- 109: under a weaker promoter to

Nos 10119- avoid

10124.

deleterious effects

Luckett et

al. 1999.

may be a strong pLlB-F- Ser Journal of

inhibitor for

HaSFTI SFTIl_nativeS 112 (SI, HaSFTI ISP 35S none pFGH060 NO Molecular

secreted

P S3) Biology

proteases 290: 525- 533.

pLlVB-F-Cl- C-

Met Arkadash et al. 2017. J. Biol.

HsTIMP TIMP2 (NtPRl 135 NtPRISP 35S terminal pFGH097 NO

alio Chem. 292: 3481-3495. SP) flag-HA

pLlVB-F-SCl- C-

Met NO Arkadash et al. 2017. J. Biol.

HsTIMP TIMP2 (native 135 HsTIMPSP 35S terminal pFGH116

alio (?) Chem. 292: 3481-3495. SP) flag-HA

pLlVB-F-SCl- C-

Met NO Arkadash et al. 2017. J. Biol.

HsTIMP TIMP2_CGCG 135 HsTIMPSP 35S terminal pFGH117

alio (?) Chem. 292: 3481-3495. P flag-HA

Arkadash

et al.

2017. J. may be a strong pLlB-F-

Met Biol. inhibitor for

HsTIMP TIMP2_NtPRl 135 NtPRISP 35S none pFGH047 YES

alio Chem. secreted

SP

292: proteases

3481-

3495.

Arkadash

et al.

2017. J. may be a strong pLlB-F-

Met YES Biol. inhibitor for

HsTIMP TIMP2_native 135 HsTIMPSP 35S none pFGH061

alio (?) Chem. secreted

SP

292: proteases

3481-

3495. may be a strong pllMB-F-35S- C-

Met inhibitor for

HsTIMP NtPRl-TIMP2- 135 HsTIMPSP 35S terminal pFGH205 NO

alio secreted 6xHis His- tag

proteases

Wingfield

et al 1999.

pllMB-F-35S- mutant version

J. Biol.

NtPRl- Met of TIMP, lacks PI

HsTIMP 135 HsTIMPSP 35S none pFGH205 NO Chem.

TIMP2_ACSCS alio y function in

274:

P animal systems.

21362-

21368.

Fluhr et al.

may be a strong 2012.

Ser/ inhibitor for

HvBSZx pLIB-F-BSZx 14 NtPRISP 35S none pFGH045 NO Physiol.

Cys secreted

Plant. 145:

proteases 95-102.

pLlVB-F-C- N. benthamiana

LB_NbCYSl 125 Cys NtPRISP 35S none pFGH109 NO

LB_NbCYSl PI pLlB-F- NO

MER411712 125 Cys NtPRISP 35S none pFGH050

MER411712 (?)

pLlVB-F-C-

NO

MER411832 MER411832_c 113 Ser NtPRISP 35S none pFGHlll

(?)

DNA

Ser/

pLlB-F-

MER411950 13 Cys/ NtPRISP 35S none pFGH051 NO

MER411950

Asp

Ser

pLlB-F- NO

MER412033 113 (SI, NtPRISP 35S none pFGH052

MER412033 (?)

S8)

van der

Linde et inhibits pLlVB-F-SC- al. 2012. endogenous

NbHvCYS6

NbHvCYS6 NbHvCYS6_gD 125 Cys 35S none pFGH114 NO Plant Cell extracellular

SP

NA Online 24: proteases in

1285- maize 1300. pLlMB-F- NtPRl- Ser/ C-

N. benthamiana

NbKl NibenlOlScfO 13 Cys/ NtPRISP 35S terminal pFGH156 NO

PI

4078g00002- Asp flag-HA

Kl-flagHA

pLlMB-F- NtPRl-

Ser/ C-

NibenlOlScfO N. benthamiana

NbK2 13 Cys/ NtPRISP 35S terminal pFGH157 NO

6424XLOC_06 PI

Asp flag-HA

4533-K2- flagHA

pLlM B-F-

NtPRl- C-

Ser/ N.

NibenlOlScf termin

NbK3 13 Cys/ NtPRISP 35S pFG H158 NO benthamiana

06424XLOC_ al flag- Asp PI

064534- K3- HA

flagHA

Ser

pLlB-F-

NbPotl 113 (SI, NtPRISP 35S none pFGH053 YES

MER412288

S8)

pLlVB-F-Cl- Ser N-

NbPotl His- 113 (SI, NtPRISP 35S terminal pFGH213 NO

MER412288 S8) His- tag

pLlVB-F-Cl- Ser C-

NbPotl MER412288- 113 (SI, NtPRISP 35S terminal pFGH213 NO

flagHA S8) flag-HA

N. benthamiana protein. High similarity to N.

Kim et al. alata protein

Ser

pLlB-F-Nb-PI- 2008. that enhances

NbPot2 120 (SI, NtPRISP 35S none pFGH046 NO

ll_dom2&3 PREP 61: RP

S8)

117-121. accumulation in rice suspension cells on co- expression

?

pLlVB-F- Kim et al. may be a strong

NbPR4 Cys NbPR4SP 35S none pFGH007 NO

NbPR4_gDNA (143) 2015. inhibitor for _native Plant J. 81: secreted signalP 81-94. proteases pLlVB-F-

? Kim et al. 2015. Plant J.81: 81-

NbPR4 NbPR4_gDNA Cys NtPRISP 35S none pFGH008 YES

(143) 94.

_NtPRlaSP

pLlVB-F-

C- NtPRlaSP- ? Kim et al. 2015. Plant J. 81:

NbPR4 Cys NtPRISP 35S terminal pFGH096 NO

NbPR4_gDNA- (143) 81-94.

flagHA

pllMB-F-35S- N-

? Kim et al. 2015. Plant J. 81:

NbPR4 NtPRl-6xHis- Cys NtPRISP 35S terminal pFGH210 NO

(143) 81-94.

NbPR4 His

N- pLlVB-F- terminal

TR007

NtPRl-flag- flag and N. benthamiana

NbSRP_LRA 14 Ser NtPRISP 35S (made by NO

NbSRP.LRA- C- PI

FGH)

3HA terminal

HA

N- terminal

pLlVB-F-flag- TR008

flag and N. benthamiana

NbSRP_LRA NbSRP.LRA- 14 Ser none 35S (made by NO

C- PI

3HA FGH)

terminal

HA

N- pLlVB-F- terminal

TR009

NtPRl-flag- flag and N. benthamiana

NbSRP_TMS 14 Ser NtPRISP 35S (made by NO

NbSRP.TMS- C- PI

FGH)

3HA terminal

HA

N- terminal

pLlVB-F-flag- TROIO

flag and N. benthamiana

NbSRP_TMS NbSRP.TMS- 14 Ser none 35S (made by NO

C- PI

3HA FGH)

terminal

HA Goulet

et al.

pLlVB-F-

Asp 2012. enhances RP SICDI_CDS_

SICDI 13 & SICDISP 35S none pFGH009 NO Plant accumulation in native

Ser Biotech plants signalP

nol J. 10:

83-94.

Goulet

et al.

pLlVB-F- Asp 2012. enhances RP

SICDI SICDI_CDS_ 13 & NtPRISP 35S none pFGHOlO NO Plant accumulation in

NtP laSP Ser Biotech plants

nol J. 10:

83-94.

Goulet

et al.

mutation

2008.

pLlB-F- appears to

Plant

SICYS8 SICYS8_8th 125 Cys NtPRISP 35S none pFGH055 NO strengthen

Physiol.

domain P2K protease

146:

inhibition

1010-

1019.

Jutras et

pLlB-F- al. 2016.

enhances RP SICYS8_8th PLOS

SICYS8 125 Cys NtPRISP 35S none pFGH054 YES accumulation in domain ONE 11:

plants native e01670

86.

dominant negative mutant of ubiquitin that

Chau et

stops the al.

prot

pLlB-F- elongation of

(1989)

easo

UbiDomNeg Nb_ubi_ none ubiquitin chains.

35S none pFGH059 NO Science

me

We reasoned

K48R 243:

TOl

that this might

1576- prevent

1584.

degradation of abundant RPs via the ubiquitin- proteasome machinery.

Overexpression of ubiquitin might enhance the function of the ubiquitin- proteasome machinery, aiding clearance

Chau et

of misfolded al.

prot proteins that pLlB-F- (1989)

easo might

Ubiquitin Nb_ubi_ none 35S none pFGH058 NO Science

me accumulate native 243:

T01 when

1576- overexpressing

1584.

RPs and thus alleviating stress that misfolded proteins inflict to cells. Also acts as a control for DomNegUb

Example 22: Conclusions

Conclusion No. 1

PI overexpression is a viable approach to resolve the degradation bottleneck, but custom- made strategies are needed for each RP.

Conclusion No. 2

Pis act in distinct ways to prevent proteolytic degradation, presumably not always by inhibiting proteases.

Sequences

Below are polynucleotide and amino acid sequences for use in accordance with the invention. HSTIMP2 AA + NTPR1 SIGNAL PEPTIDE AA

MGFVLFSQLPSFLLVSTLLLFLVISHSCRAGGCSCSPVHPQQAFCNADWIRAKAVSEKEVD SGNDIYGNPIKRIQYEIKQIKMFKGPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDG KMHITLCDFIVPWDTLSTTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEK NINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP [SEQ ID NO: 1]

HSTIMP2 AA (NO SIGNAL PEPTIDE)

CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPEKDIEFI YTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLSTTQKKSLNHRYQM GCECKITRCPMIPCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPK QEFLDIEDP [SEQ ID NO: 2]

CSCSP [SEQ ID NO: 3]

APSSAVC [SEQ ID NO: 4]

SCAWYRGAA [SEQ ID NO: 5]

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP [SEQ ID NO: 6]

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[SEQ ID NO: 7]

CSCSPVHPQQAFCNADVVIRAKAVSEKE [SEQ ID NO: 8]

HSTIMP2 DNA CODING SEQUENCE

TGTTCTTGCTCTCCTGTTCATCCTCAGCAGGCTTTCTGCAATGCTGATGTGGTGATTAG

GGCTAAGGCTGTGAGCGAGAAAGAAGTGGATAGCGGTAACGATATCTACGGTAACCCT

ATCAAGAGGATCCAGTACGAGATCAAGCAGATCAAGATGTTCAAGGGTCCTGAGAAGG

ATATCGAGTTTATCTACACCGCTCCTAGCTCTGCTGTTTGCGGTGTTTCTCTTGATGTGG

GTGGTAAGAAAGAGTACCTGATCGCTGGTAAGGCTGAGGGTGATGGTAAGATGCACAT

TACCCTGTGCGATTTCATCGTGCCTTGGGATACCCTTTCAACCACTCAGAAGAAGTCCC

TGAACCACAGGTATCAGATGGGTTGCGAGTGCAAGATTACCAGGTGCCCTATGATCCC

TTGCTACATCTCTTCACCTGATGAGTGCCTGTGGATGGATTGGGTTACCGAGAAGAACA

TCAACGGTCACCAGGCTAAGTTCTTCGCTTGCATCAAGAGGTCCGATGGTTCTTGCGCT

TGGTATAGAGGTGCTGCTCCTCCTAAGCAAGAGTTCCTTGATATCGAGGATCCTTAG

[SEQ ID NO: 9]

HsTIMP2 DNA CODING SEQUENCE + NTPR1 SIGNAL PEPTIDE (UNDERLINED)

ATGGGTGCTGCTGCTAGGACTCTTAGGCTTGCTCTTGGTCTGCTTCTTCTGGCTACTCT TCTTAGGCCTGCTGATGCTTGTTCTTGCTCTCCTGTTCATCCTCAGCAGGCTTTCTGCA ATGCTGATGTGGTGATTAGGGCTAAGGCTGTGAGCGAGAAAGAAGTGGATAGCGGTAA

CGATATCTACGGTAACCCTATCAAGAGGATCCAGTACGAGATCAAGCAGATCAAGATGT

TCAAGGGTCCTGAGAAGGATATCGAGTTTATCTACACCGCTCCTAGCTCTGCTGTTTGC

GGTGTTTCTCTTGATGTGGGTGGTAAGAAAGAGTACCTGATCGCTGGTAAGGCTGAGG

GTGATGGTAAGATGCACATTACCCTGTGCGATTTCATCGTGCCTTGGGATACCCTTTCA

ACCACTCAGAAGAAGTCCCTGAACCACAGGTATCAGATGGGTTGCGAGTGCAAGATTA

CCAGGTGCCCTATGATCCCTTGCTACATCTCTTCACCTGATGAGTGCCTGTGGATGGAT

TGGGTTACCGAGAAGAACATCAACGGTCACCAGGCTAAGTTCTTCGCTTGCATCAAGA

GGTCCGATGGTTCTTGCGCTTGGTATAGAGGTGCTGCTCCTCCTAAGCAAGAGTTCCTT

GATATCGAGGATCCTTAG [SEQ ID NO: 10]

TVRIVDQC [SEQ ID NO: 11]

NGGLDLD [SEQ ID NO: 12]

CGRCLRVTNT [SEQ ID NO: 13]

LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]

AGGQSATNVRSTYHLYNPQNINWDL [SEQ ID NO: 15]

NBPR4 AA

QSATNVRSTYHLYNPQNINWDLRAASAFCTTWDADKPLTWRQKYGWTAFCDAAGPQGQD SCGRCLRVTNTGTGTQTTVRIVDQCRNGGLDLDVNVFNQLDTNGVGYQQGHLIVNYEFINC DD [SEQ ID NO: 16]

NBPR4 DNA CODING SEQUENCE + INTRON (UNDERLINED)

CAGAGCGCTACAAACGTGAGATCAACGTATCATTTATACAACCCACAGAACATTAACTG

GGATTTGAGAGCAGCAAGTGCTTTCTGCACTACTTGGGATGCCGACAAGCCTCTCACG

TGGCGTCAGAAATATGGCTGGACTGCTTTCTGTGATGCTGCTGGACCTCAAGGCCAAG

ATTCCTGTGGTAGATGCTTGAGGGTACGACATCTTCAATTTTTTGTTACTATATCAATTTT

GTCATGAAAGATTTACCTATTGATGTAGGTCAACTTTTACCTAATGGTATATATGTCGGT

GCACATAATTTAAACTCTATAGTTTGTTTTAGAGTTTAACTTCTATATACTTGTCGGTATA

AAAGCATTTTGCACTATCAAATGTTTACTATAAGTAAATTATTTGAAAGAGAAAATGCCGT

AAGTAGCAAAGTAACTAAAAATACTGAATACCTGGATATGTATTAATGGTAAAGCTAAAC

GCAGGTGACGAACACAGGAACAGGAACTCAAACAACAGTGAGAATAGTAGATCAATGC

AGAAATGGAGGGCTTGATTTGGATGTAAACGTCTTTAACCAATTGGACACAAATGGAGT

GGGCTATCAGCAAGGCCACCTTATTGTCAACTATGAATTTATCAACTGCGATGACTAAG

CTT [SEQ ID NO: 17] NBPR4 DNA CODING SEQUENCE + INTRON (ITALIC) + NTPR1 SIGNAL PEPTIDE (UNDERLINED)

ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTAT

TCCTAGTAATATCCCACTCTTGCCGTGCACAGAGCGCTACAAACGTGAGATCAACGTAT

CATTTATACAACCCACAGAACATTAACTGGGATTTGAGAGCAGCAAGTGCTTTCTGCAC

TACTTGGGATGCCGACAAGCCTCTCACGTGGCGTCAGAAATATGGCTGGACTGCTTTC

TGTGATGCTGCTGGACCTCAAGGCCAAGATTCCTGTGGTAGATGCTTGAGGGLACG/AC

A TCTTCAA TTTTTTGTTACTA TA TCAA TTTTGTCATGAAAGA TTTACCTATTGA TGTAGGT

CAACTTTTACCTAATGGTATATATGTCGGTGCACATAATTTAAACTCTATAGTTTGTTTTA

GAGTTTAACTTCTATATACTTGTCGGTATAAAAGCATTTTGCACTATCAAATGTTTACTAT

AAGTAAATTATTTGAAAGAGAAAATGCCGTAAGTAGCAAAGTAACTAAAAATACTGAATA

CCTGGATATGTATTAATGGTAAAGCTAAACGCAGGJGACGAACACAGGAACAGGAACJC

AAACAACAGTGAGAATAGTAGATCAATGCAGAAATGGAGGGCTTGATTTGGATGTAAAC

GTCTTTAACCAATTGGACACAAATGGAGTGGGCTATCAGCAAGGCCACCTTATTGTCAA

CTATGAATTTATCAACTGCGATGACTAAGCTT [SEQ ID NO: 18]

KX1X2WPELVG [SEQ ID NO: 19]

KHLWPELVG [SEQ ID NO: 20]

NWEFIVIITPTVG [SEQ ID NO: 21]

GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]

NBPOT1 AA

KHLWPELVGMPGKTAKEIIEKENPLVKVQFLFPGMPKPLDLVCGRVIVWNWEFIVIITPTVG

[SEQ ID NO: 23]

NBPOT1 NT

GGTAAGTTTCTGCTTCTACCTTTGATATATATATAATAATTATCATTAATTAGTAGTAATAT

AATATTTCAAATATTTTTTTCAAAATAAAAGAATGTAGTATATAGCAATTGCTTTTCTGTAG

TTTATAAGTGTGTATATTTTAATTTATAACTTTTCTAATATATGACCAAAATTTGTTGATGT

GCAGGAGGTAAGCATTTATGGCCTGAACTTGTGGGAATGCCAGGAAAGACTGCTAAGG

AAATAATTGAGAAAGAAAATCCCTTGGTCAAAGTTCAATTTTTGTTCCCTGGAATGCCTA

AACCATTGGATTTAGTTTGTGGTCGAGTTATTGTTGTTGTTAACTGGGAATTCATCGTTA

TAATTACTCCCACAGTGGGTTAA [SEQ ID NO: 24]

NBPOT1 NT + NTPR1 SIGNAL PEPTIDE NT (UNDERLINED)

ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTAT TCCTAGTAATATCCCACTCTTGCCGTGCAGGTAAGTTTCTGCTTCTACCTTTGATATATA TATAATAATTATCATTAATTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGA

ATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAACTT

TTCTAATATATGACCAAAATTTGTTGATGTGCAGGAGGTAAGCATTTATGGCCTGAACTT

GTGGGAATGCCAGGAAAGACTGCTAAGGAAATAATTGAGAAAGAAAATCCCTTGGTCAA

AGTTCAATTTTTGTTCCCTGGAATGCCTAAACCATTGGATTTAGTTTGTGGTCGAGTTAT

TGTTGTTGTTAACTGGGAATTCATCGTTATAATTACTCCCACAGTGGGTTAA [SEQ ID

NO: 25]

NBPR4 AA + NTPR1 SIGNAL PEPTIDE AA

MGFVLFSQLPSFLLVSTLLLFLVISHSCRAGGQSATNVRSTYHLYNPQNINWDLRAASAFCT TWDADKPLTWRQKYGWTAFCDAAGPQGQDSCGRCLRVTNTGTGTQTTVRIVDQCRNGG LDLDVNVFNQLDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 26]

NBPOT1 AA + NTPR1 SIGNAL PEPTIDE AA

MGFVLFSQLPSFLLVSTLLLFLVISHSCRAGGKHLWPELVGMPGKTAKEIIEKENPLVKVQFL FPGMPKPLDLVCGRVIVVVNWEFIVIITPTVG [SEQ ID NO: 27]

NTPR1 SIGNAL PEPTIDE AA (GLY-GLY LINKER UNDERLINED)

MGFVLFSQLPSFLLVSTLLLFLVISHSCRAGG [SEQ ID NO: 28]

NTPR1 SP DNA SEQUENCE USED TO REPLACE NATIVE SIGNAL PEPTIDES (CDS UNDERLINED, INTRON IN BLACK, GLY-GLY LINKER IN ITALICS)

ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTAT TCCTAGTAATATCCCACTCTTGCCGTGCAGGTAAGTTTCTGCTTCTACCTTTGATATATA TATAATAATTATCATTAATTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGA ATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAACTT TTCTA ATAT ATG ACC A AA ATTTGTTG ATGTG C A G GA G G 7 [S EQ ID NO: 29]

HSTIMP2 NATIVE PROTEIN (NATIVE SIGNAL PEPTIDE UNDERLINED, N-TERMINAL CYS RESIDUES IN BOLD)

GAAARTLRLALGLLLLATLLRPADACSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYG NPIKRIQYEIKQIKMFKGPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLC DFIVPWDTLSTTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKNINGHQA KFFACI KRSDGSCAWYRGAAPPKQEFLDI EDP [SEQ ID NO: 30]

HSTIMP2 EXPRESSION CONSTRUCT (35S: NTPR1 SP:HSTIMP2)

GTCAACATGGTGGAGCACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTC AGAAGATCAAAGGGCTATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCG GATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGC

TCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATCTCTCTGCCGA

CAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAGGTT

CCAACCACGTCTACAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCT

GGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGATCAAAGGGCTATTGAGACTT

TTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCAC

TTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAA

AGGAAAGGCTATCATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCA

CCCACGAGGAGCATCGTGGAAAAAGAAGAGGTTCCAACCACGTCTACAAAGCAAGTGG

ATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAA

GACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTCGAGTATAAG

AGCTCATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAATTACATTT

ACAATTATCGATACGAATGGGTGCTGCTGCTAGGACTCTTAGGCTTGCTCTTGGTCTGC

TTCTTCTGGCTACTCTTCTTAGGCCTGCTGATGCTTGTTCTTGCTCTCCTGTTCATCCTC

AGCAGGCTTTCTGCAATGCTGATGTGGTGATTAGGGCTAAGGCTGTGAGCGAGAAAGA

AGTGGATAGCGGTAACGATATCTACGGTAACCCTATCAAGAGGATCCAGTACGAGATCA

AGCAGATCAAGATGTTCAAGGGTCCTGAGAAGGATATCGAGTTTATCTACACCGCTCCT

AGCTCTGCTGTTTGCGGTGTTTCTCTTGATGTGGGTGGTAAGAAAGAGTACCTGATCGC

TGGTAAGGCTGAGGGTGATGGTAAGATGCACATTACCCTGTGCGATTTCATCGTGCCTT

GGGATACCCTTTCAACCACTCAGAAGAAGTCCCTGAACCACAGGTATCAGATGGGTTG

CGAGTGCAAGATTACCAGGTGCCCTATGATCCCTTGCTACATCTCTTCACCTGATGAGT

GCCTGTGGATGGATTGGGTTACCGAGAAGAACATCAACGGTCACCAGGCTAAGTTCTT

CGCTTGCATCAAGAGGTCCGATGGTTCTTGCGCTTGGTATAGAGGTGCTGCTCCTCCT

AAGCAAGAGTTCCTTGATATCGAGGATCCTTAGGCTTCTCTAGCTAGAGTCGATCGACA

AGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCC

TATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGT

AAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAG

AT [SEQ ID NO: 31]

NBPR41 NATIVE PROTEIN (NATIVE SIGNAL PEPTIDE UNDERLINED)

MERVNNYYKLCMELFIMSMMVAMAAAQSATNVRSTYH LYN PQN I N WDLRAASAFCTTWDA DKPLTWRQKYGWTAFCDAAGPQGQDSCGRCLRVTNTGTGTQTTVRIVDQCRNGGLDLDV NVFNQLDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 32]

NBPR4 EXPRESSION CONSTRUCT (35S PROMOTER AND TERMINATOR + NTPR1 SIGNAL PEPTIDE UNDERLINED, CODING SEQUENCE IN ITALICS) GTCAACATGGTGGAGCACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTC

AGAAGATCAAAGGGCTATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCG

GATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGC

TCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATCTCTCTGCCGA

CAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAGGTT

CCAACCACGTCTACAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCT

GGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGATCAAAGGGCTATTGAGACTT

TTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCAC

TTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAA

AGGAAAGGCTATCATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCA

CCCACGAGGAGCATCGTGGAAAAAGAAGAGGTTCCAACCACGTCTACAAAGCAAGTGG

ATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAA

GACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTCGAGTATAAG

AGCTCATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAATTACATTT

ACAATTATCGATACGAATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTC

TCTACACTTCTCTTATTCCTAGTAATATCCCACTCTTGCCGTGCAC \G \GCGCLAC \ \ \C

G TGA GA TCAA CGTA TCA TTTATACAACCCACAGAACATTAACTGGGATTTGAGAGCAGC

AAGTGCTTTCTGCACTACTTGGGATGCCGACAAGCCTCTCACGTGGCGTCAGAAATATG

GCTGGACTGCTTTCTGTGATGCTGCTGGACCTCAAGGCCAAGATTCCTGTGGTAGATG

C TTGA G GGTACG ACATCTTC AATTTTTTGTTACTATATC AATTTTGTC ATG AAAGATTTAC

CTATTGATGTAGGTCAACTTTTACCTAATGGTATATATGTCGGTGCACATAATTTAAACT

CTATAGTTTGTTTTAGAGTTTAACTTCTATATACTTGTCGGTATAAAAGCATTTTGCACTA

TCAAATGTTTACTATAAGTAAATTATTTGAAAGAGAAAATGCCGTAAGTAGCAAAGTAAC

TAAAAATACTGAATACCTGGATATGTATTAATGGTAAAGCTAAACGCAGGTGACGAAC/A

CAGGAACAGGAACTCAAACAACAGTGAGAATAGTAGATCAATGCAGAAATGGAGGGCT

TGATTTGGATGTAAACGTCTTTAACCAATTGGACACAAATGGAGTGGGCTATCAGCAAG

GCCACCTTATTG TCAACTA TGAA TTTATCAACTGCGA TGACTAAG CTTCJCJAGCJAGAG

TCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAA

TTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTAT

TTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACT

AAAATCCAGAT [SEQ ID NO: 33]

NBPOT1 DNA CODING SEQUENCE

AAGCATTTATGGCCTGAACTTGTGGGAATGCCAGGAAAGACTGCTAAGGAAATAATTGA GAAAGAAAATCCCTTGGTCAAAGTTCAATTTTTGTTCCCTGGAATGCCTAAACCATTGGA TTTAGTTTGTGGTCGAGTTATTGTTGTTGTTAACTGGGAATTCATCGTTATAATTACTCCC ACAGTGGGTTAA [SEQ ID NO: 34] NBPOT1 EXPRESSION CONSTRUCT (35S PROMOTER AND TERMINATOR, NTPR1 SIGNAL PEPTIDE (UNDERLINED), NBPOT1 CODING SEQUENCE (ITALICS))

GTCAACATGGTGGAGCACGACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTC

AGAAGATCAAAGGGCTATTGAGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCG

GATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGC

TCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATCTCTCTGCCGA

CAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGAGGTT

CCAACCACGTCTACAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCT

GGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGATCAAAGGGCTATTGAGACTT

TTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCAC

TTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAA

AGGAAAGGCTATCATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCA

CCCACGAGGAGCATCGTGGAAAAAGAAGAGGTTCCAACCACGTCTACAAAGCAAGTGG

ATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAA

GACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTCGAGTATAAG

AGCTCATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAATTACATTT

ACAATTATCGATACAATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCT

CTACACTTCTCTTATTCCTAGTAATATCCCACTCTTGCCGTGCAGGTAAGTTTCTGCTTC

TACCTTTGATATATATATAATAATTATCATTAATTAGTAGTAATATAATATTTCAAATATTTT

TTTCAAAATAAAAGAATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATA

TTTTAATTTATAACTTTTCTAATATATGACCAAAATTTGTTGATGTGCAGGAGGT \ \GC \7

TTA TGGCCTGAACTTGTGGGAA TGCCAGGAAAGACTGCTAAGGAAA TAA TTGAGAAAGA

AAA TCCCTTGGTCAAAGTTCAA TTTTTGTTCCCTGGAATGCCTAAACCA TTGGA TTTAGT

TTGTGGTCGAGTTATTGTTGTTGTTAACTGGGAATTCATCGTTATAATTACTCCCACAGT

GGGTTAAGCTTCTCTAGCTAGAGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTG

TGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGC

ATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAA

TTCCTAAAACCAAAATCCAGTACTAAAATCCAGAT [SEQ ID NO: 35]

The following numbered paragraph are not the claims but statements of the invention which may in some instances duplicate previous statements herein.

1. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 1 or a sequence of at least 63% identity thereto or a functional fragment thereof.

2. A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more

recombinant proteins and Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide in the apoplast of the plant or plant part.

3. A method of paragraph 2, wherein the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58% identity thereto or a functional fragment thereof.

4. A method of paragraphs 2 or 3, wherein the HsTIMP2 polypeptide comprises a signal peptide capable of directing HsTIMP2 polypeptide accumulation in the apoplast of a plant.

5. A method of paragraph 4, wherein the HsTIMP2 comprises a polypeptide sequence of SEQ ID NO: 1.

6. A method of any preceding paragraph, wherein the HsTIMP2 polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or

b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or

c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or

d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or

e. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQ EFLDIEDP [SEQ ID NO: 7]; and/or

f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO:

8].

7. A method of any preceding paragraph, wherein the HsTIMP2 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof.

8. A method of any preceding paragraph, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ I D NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ I D NO: 10. A method of any preceding paragraph, wherein the method further comprises increasing the level or expression of NbPR4 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. A method of paragraph 9, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ I D NO: 1 1 ]; and/or b. the amino acid motif NGGLDLD [SEQ I D NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFI NCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ I D NO: 15]; and/or f. the polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or functional fragment thereof. A method of paragraphs 9 or 10, wherein the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ I D NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ I D NO: 18. A method of any preceding paragraph, wherein the method further comprises increasing the level or expression of NbPotI polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. A method of paragraph 12, wherein the NbPotI polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ I D NO: 19]; wherein X^ is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or

Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ I D NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]; and/or e. the polypeptide sequence [SEQ ID NO: 23] or a sequence of at least 50% identity thereto or a functional fragment thereof. A method of paragraph 12 or 13, wherein the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25. A method of any of paragraphs 8 to 14, wherein said plant or plant part is

transformed with a multiplicity of said polynucleotides. A method of any of paragrahs 8 to 15, wherein the plant or plant part is transiently transformed with one or more of said polynucleotides. A method of any of paragraphs 8 to 15, wherein the plant or plant part has one or more of said polynucleotides stably incorporated into its genome. A method of any of paragraphs 8 to 15, wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part. A method of any preceding paragraph, wherein the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. A method of any preceding paragraph, wherein the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal). A method of any preceding paragraph, wherein the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. A method of preceding paragraph, wherein the polynucleotides encoding the

HsTIMP2 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. A method of any of paragraphs 15 to 22, wherein the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. A method of any preceding paragraph, wherein the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant. A method of paragraph 24, wherein the plant or plant part is of the genus Nicotiana. A method of paragraph 25, wherein the plant or plant part is a N. benthamiana plant or plant part. A method of any preceding paragraph, wherein said plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells. A modified plant or plant part genetically engineered to be capable of expressing Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide in the apoplast of the plant or plant part.

A modified plant or plant part of paragraph 28, wherein the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58% identity thereto or a functional fragment thereof. A modified plant or plant part of paragraph 28 or 29, wherein the HsTIMP2 polypeptide comprises a signal peptide capable of directing HsTIMP2 polypeptide accumulation in the apoplast of a plant. A modified plant or plant part of paragraph 30, wherein the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: 1. A modified plant or plant part of paragraphs 28 to 31 , wherein the HsTIMP2 polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or

b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or

c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or

d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or

e. the amino acid motif

f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO:

8].

A modified plant or plant part of any of paragraphs 28 to 32, wherein the HsTIMP2 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof. A modified plant or plant part of any of paragraphs 28 to 33, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10. A modified plant or plant part of any of paragraphs 28 to 34, wherein the modified plant or plant part is genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part. A modified plant or plant part of paragraph 35, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]; and/or f. the polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof. A modified plant or plant part of paragraph 35 or 36, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18. A modified plant or plant part of any of paragraphs 28 to 37, wherein the modified plant or plant part is genetically engineered to be capable of expressing NbPotl polypeptide in the apoplast of the plant or plant part. A modified plant or plant part of paragraph 38, wherein the NbPotl polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or

Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]; and/or e. the polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof. A modified plant or plant part of paragraph 39 or 40, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25. A modified plant or plant part of any of paragraphs 34 to 40, wherein said modified plant or plant part is transformed with a multiplicity of said polynucleotides. A modified plant or plant part of any of paragraphs 34 to 41 , wherein said modified plant or plant part is transiently transformed with one or more of said polynucleotides. A modified plant or plant part of any of paragraphs 34 to 42, wherein said modified plant or plant part has one or more of said polynucleotides stably incorporated into its genome. A modified plant or plant part of any of paragraphs 34 to 43, wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part. A modified plant or plant part of any of paragraphs 28 to 44, wherein the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. A modified plant or plant part of any of paragraphs 28 to 45, wherein the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha- 1 ,3-galactose (a-Gal). A modified plant or plant part of any of paragraphs 28 to 46, wherein said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part. A modified plant or plant part of any of paragraphs 34 to 47, wherein the

polynucleotides encoding the HsTIMP2 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. A modified plant or plant part of any of paragraphs 41 to 48, wherein the

polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector. A modified plant or plant part of any of paragraphs 28 to 49, wherein the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant. A modified plant or plant part of paragraph 50, wherein the modified plant or plant part is of the genus Nicotiana. A modified plant or plant part of paragraph 51 , wherein the modified plant or plant part is, or is of a N. benthamiana plant. A modified plant or plant part of any of paragraphs 28 to 52, wherein said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells. Use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58% identity thereto or a functional fragment thereof or a sequence of SEQ ID NO: 1 or a sequence of at least 63% identity thereto or a functional fragment thereof. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 26 or a sequence of at least 63% identity thereto or a functional fragment thereof.

A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more

recombinant proteins and NbPR4 polypeptide in the apoplast of the plant or plant part. A method of paragraph 56, wherein the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof. A method of paragraph 56 or 57, wherein the NbPR4 polypeptide comprises a signal peptide capable of directing NbPR4 polypeptide accumulation in the apoplast of a plant. A method of paragraph 58, wherein the NbPR4 polypeptide comprises a polypeptide sequence of SEQ ID NO: 26. A method of any of paragraphs 56 to 59, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]. A method of any paragraphs 56 to 60, wherein the NbPR4 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof. A method of any of paragraphs 56 to 61 , wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18. A method of any of paragraphs 56 to 62, wherein the method further comprises increasing the level or expression of HsTIMP2 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. A method of paragraph 63, wherein the HsTIMP2 polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or

b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or

c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or

d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or

e. the amino acid motif

f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO:

8]; and/or

g. a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58%

identity thereto or a functional fragment thereof. A method of paragraph 63 or 64, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10. 66. A method of any of paragraphs 56 to 65, wherein the method further comprises increasing the level or expression of NbPotl polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

67. A method of paragraph 66, wherein the NbPotl polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]; and/or e. the polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

68. A method of paragraph 66 or 67, wherein the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25.

69. A method of paragraphs 62 to 68, wherein said plant or plant part is transformed with a multiplicity of said polynucleotides.

70. A method of any of paragraphs 62 to 69, wherein the plant or plant part is transiently transformed with one or more of said polynucleotides.

71. A method of any of paragraphs 62 to 70, wherein the plant or plant part has one or more of said polynucleotides stably incorporated into its genome.

72. A method ofany of paragraphs 62 to 71 , wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

73. A method of any of paragraphs 56 to 72, wherein the plant or plant part has an

increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

74. A method of any of paragraphs 56 to 73, wherein the one or more recombinant

proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal).

75. A method of any of paragraphs 56 to 74, wherein the plant or plant part has an

increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

76. A method of any of paragraphs 56 to 75, wherein the polynucleotides encoding the NbPR4 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

77. A method of any of paragraphs 69 to 76, wherein the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

78. A method of any of paragraphs 56 to 77, wherein the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant.

79. A method of paragraph 78, wherein the plant or plant part is of the genus Nicotiana.

80. A method of paragraph 79, wherein the plant or plant part is a N. benthamiana plant or plant part.

81. A method of any of paragraphs 56 to 80, wherein said plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

82. A modified plant or plant part genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part.

83. A modified plant or plant part of paragraph 82, wherein the NbPR4 polypeptide

comprises a polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof. 84. A modified plant or plant part of paragraph 82 or 83, wherein the NbPR4 polypeptide comprises a signal peptide capable of directing NbPR4 polypeptide accumulation in the apoplast of a plant.

85. A modified plant or plant part of paragraph 84, wherein the NbPR4 polypeptide

comprises a polypeptide sequence of SEQ ID NO: 26.

86. A modified plant or plant part of any of paragraphs 82 to 85, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15].

87. A modified plant or plant part of any of paragraphs 82 to 86, wherein the NbPR4 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof.

88. A modified plant or plant part of any of cparagraphs 82 to 87, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18.

89. A modified plant or plant part of any of paragraphs 82 to 88, wherein the modified plant or plant part is genetically engineered to be capable of expressing HsTIMP2 polypeptide in the apoplast of the plant or plant part.

90. A modified plant or plant part of paragraph 89, wherein the HsTIMP2 polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or e. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQ EFLDIEDP [SEQ ID NO: 7]; and/or f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO:

8]; and/or g. a polypeptide sequence of SEQ ID NO:2 [HsTIMP2 AA] or a sequence of at least 58% identity thereto or a functional fragment thereof.

91. A modified plant or plant part of paragraph 89 or paragraph 90, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10.

92. A modified plant or plant part of any of paragraphs 82 to 91 , wherein the modified plant or plant part is genetically engineered to be capable of expressing NbPotl polypeptide in the apoplast of the plant or plant part.

93. A modified plant or plant part of paragraph 92, wherein the NbPotl polypeptide

comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]; and/or e. the polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

94. A modified plant or plant part of paragraph 92 or 93, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25.

95. A modified plant or plant part of any of paragraphs 88 to 94, wherein said modified plant or plant part is transformed with a multiplicity of said polynucleotides.

96. A modified plant or plant part of any of paragraphs 88 to 95, wherein said modified plant or plant part is transiently transformed with one or more of said polynucleotides.

97. A modified plant or plant part of any of paragraph 88 to 96, wherein said modified plant or plant part has one or more of said polynucleotides stably incorporated into its genome.

98. A modified plant or plant part of any of paragraphs 89 to 97, wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

99. A modified plant or plant part of any of paragraphs 90 to 98, wherein the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

100. A modified plant or plant part of any of paragraphs 82 to 99, wherein the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or

Galactose-alpha-1 ,3-galactose (a-Gal).

101. A modified plant or plant part of any of paragraphs 82 to 100, wherein said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

102. A modified plant or plant part of any of paragraphs 88 to 101 , wherein the polynucleotides encoding the NbPR4 polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

103. A modified plant or plant part of any of paragraphs 95 to 102, wherein the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

104. A modified plant or plant part of any of paragraphs 82 to 103, wherein the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant.

105. A modified plant or plant part of paragraph 104, wherein the modified plant or plant part is of the genus Nicotiana.

106. A modified plant or plant part of paragraph 105, wherein the modified plant or plant part is, or is of a N. benthamiana plant.

107. A modified plant or plant part of any of paragraphs 82 to 106, wherein said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

108. Use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof or a sequence of SEQ ID NO: 26 or a sequence of at least 63% identity thereto or a functional fragment thereof.

109. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 27 or a sequence of at least 50% identity thereto or a functional fragment thereof.

1 10. A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and NbPotI polypeptide in the apoplast of the plant or plant part.

1 11. A method of paragraph 110, wherein the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

1 12. A method of paragraph 110 or claim 1 11 , wherein the NbPotI polypeptide comprises a signal peptide capable of directing NbPotI polypeptide accumulation in the apoplast of a plant.

1 13. A method of paragraph 112, wherein the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 27. . A method of any of paragraphs 1 10 to 113, wherein the NbPotl polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or

Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22]. . A method of any of paragraphs 1 10 to 114, wherein the NbPotl polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof. . A method of any of paragraphs 1 10 to 115, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25. . A method of any of paragraphs 1 10 to 116, wherein the method further comprises increasing the level or expression of HsTIMP2 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. . A method of paragraph 117, wherein the HsTIMP2 polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or

b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or

c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or

d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or

e. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQ EFLDIEDP [SEQ ID NO: 7]; and/or f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO: 8]; and/or

g. a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58%

identity thereto or a functional fragment thereof. . A method of paragraph 117 or 118, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10. . A method of any of paragraphs 1 10 to 120, wherein the method further comprises increasing the level or expression of NbPR4 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part. . A method of paragraph 120, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]; and/or f. the polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof. . A method of paragraph 120 or 121 , wherein the method comprises transforming the plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18. 123. A method of paragraphs 116 to 122, wherein said plant or plant part is transformed with a multiplicity of said polynucleotides.

124. A method of any of paragraphs 1 16 to 123, wherein the plant or plant part is transiently transformed with one or more of said polynucleotides.

125. A method of any of paragraphs 1 16 to 124, wherein the plant or plant part has one or more of said polynucleotides stably incorporated into its genome.

126. A method of any of paragraphs 1 16 to 125, wherein one or more of the

polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

127. A method of any of paragraphs 1 10 to 126, wherein the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part.

128. A method of any of paragraphs 1 10 to 127, wherein the one or more

recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha- 1 ,3-galactose (a-Gal).

129. A method of any of paragraphs 1 10 to 128, wherein the plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotI in the apoplast of the plant or plant part.

130. A method of any of paragraphs 1 10 to 129, wherein the polynucleotides

encoding the NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

131. A method of any of paragraphs 123 to 130, wherein the polynucleotides

encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotI polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

132. A method of any of paragraphs 1 10 to 131 , wherein the plant is a

dicotyledonous plant, or the plant part is of a dicotyledonous plant.

133. A method of paragraph 132, wherein the plant or plant part is of the genus Nicotiana. 134. A method of paragraph 133, wherein the plant or plant part is a N.

benthamiana plant or plant part.

135. A method of any of paragraphs 1 10 to 134, wherein said plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

136. A modified plant or plant part genetically engineered to be capable of

expressing NbPotl polypeptide in the apoplast of the plant or plant part.

137. A modified plant or plant part of paragraph 136, wherein the NbPotl

polypeptide comprises a polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

138. A modified plant or plant part of paragraph 136 or 137, wherein the NbPotl polypeptide comprises a signal peptide capable of directing NbPotl polypeptide accumulation in the apoplast of a plant.

139. A modified plant or plant part of paragraph 138, wherein the NbPotl

polypeptide comprises a polypeptide sequence of SEQ ID NO: 27.

140. A modified plant or plant part of any of paragraphs 136 to 139, wherein the NbPotl polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22].

141. A modified plant or plant part of any of paragraphs 136 to 140, wherein the NbPotl polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof.

142. A modified plant or plant part of any paragraph 136 to 141 , wherein the

modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 24.

143. A modified plant or plant part of any of paragraphs 136 to 142, wherein the modified plant or plant part is genetically engineered to be capable of expressing HsTIMP2 polypeptide in the apoplast of the plant or plant part.

144. A modified plant or plant part of paragraph 143, wherein the HsTIMP2

polypeptide comprises; a. the amino acid motif CSCSP [SEQ ID NO: 3]; and/or b. the amino acid motif APSSAVC [SEQ ID NO: 4]; and/or c. the amino acid motif SCAWYRGAA [SEQ ID NO: 5]; and/or d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or e. the amino acid motif

8]; and/or g. a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58%

identity thereto or a functional fragment thereof.

145. A modified plant or plant part of paragraph 143 or claim 144, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10.

146. A modified plant or plant part of any of paragraphs 136 to 145, wherein the modified plant or plant part is genetically engineered to be capable of expressing NbPR4 polypeptide in the apoplast of the plant or plant part. 147. A modified plant or plant part of paragraph 146, wherein the NbPR4 polypeptide comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 11]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I N WDL [SEQ ID NO: 15]; and/or f. the polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof.

148. A modified plant or plant part of paragraph 146 or 147, wherein the modified plant or plant part is transformed with a polynucleotide comprising; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18.

149. A modified plant or plant part of any of paragraphs 142 to 148, wherein said modified plant or plant part is transformed with a multiplicity of said polynucleotides.

150. A modified plant or plant part of any of paragraphs 142 to 149, wherein said modified plant or plant part is transiently transformed with one or more of said polynucleotides.

151. A modified plant or plant part of any of paragraphs 142 to 150, wherein said modified plant or plant part has one or more of said polynucleotides stably

incorporated into its genome.

152. A modified plant or plant part of any of paragraphs 142 to 151 , wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

153. A modified plant or plant part of any of paragraphs 136 to 152, wherein the modified plant or plant part has an increased level of the one or more recombinant proteins compared to an equivalent plant or plant part, which is modified to express the one or more recombinant proteins but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

154. A modified plant or plant part of any of paragraphs 136 to 153, wherein the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha-1 ,3-galactose (a-Gal).

155. A modified plant or plant part of any of paragraphs 136 to 154, wherein said modified plant or plant part has an increased level of recombinant VCR01 and/or EPO and/or a-Gal compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

156. A modified plant or plant part of any of paragraphs 142 to 155, wherein the polynucleotides encoding the NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

157. A modified plant or plant part of any of paragraphs 149 to 156, wherein the polynucleotides encoding the HsTIMP2 polypeptide, NbPR4 polypeptide, NbPotl polypeptide and a polynucleotide sequence encoding the one or more recombinant proteins are comprised in the same expression vector.

158. A modified plant or plant part of any of paragraphs 136 to 157, wherein the plant is a dicotyledonous plant, or the plant part is of a dicotyledonous plant.

159. A modified plant or plant part of paragraph 158, wherein the modified plant or plant part is of the genus Nicotiana.

160. A modified plant or plant part as claimed in claim 159, wherein the modified plant or plant part is, or is of a N. benthamiana plant.

161. A modified plant or plant part of any of paragraphs 136 to 160, wherein said modified plant or plant part is selected from any of whole plants, leaves, stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs, husks, stalks, root tips, anthers, pericarp, silk, tissue or cells.

162. Use of a protease inhibitor in improving the level of recombinant protein in a plant or plant part, wherein the protease inhibitor comprises a polypeptide sequence of SEQ ID NO: or a sequence of at least 50% identity thereto or a functional fragment thereof; or a sequence of SEQ ID NO: 27 or a sequence of at least 50% identity thereto or a functional fragment thereof.

Claims

A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and Tissue Inhibitor of Metalloproteinase 2

(HsTIMP2)

polypeptide in the apoplast of the plant or plant part.

A method as claimed in claim 1 , wherein the HsTIMP2 polypeptide comprises a polypeptide sequence of SEQ ID NO: 2 or a sequence of at least 58% identity thereto or a functional fragment thereof.

A method as claimed in claim 1 or claim 2, wherein the HsTIMP2 polypeptide comprises a signal peptide capable of directing HsTIMP2 polypeptide accumulation in the apoplast of a plant; optionally wherein the HsTIMP2 comprises a polypeptide sequence of SEQ ID NO: 1.

A method as claimed in any preceding claim, wherein the HsTIMP2 polypeptide comprises;

a. the amino acid motif CSCSP

[SEQ ID NO: 3]; and/or

b. the amino acid motif APSSAVC

[SEQ ID NO: 4]; and/or

c. the amino acid motif SCAWYRGAA

[SEQ ID NO: 5]; and/or

d. the amino acid motif

PCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPP

[SEQ ID NO: 6]; and/or

e. the amino acid motif

f. the amino acid motif CSCSPVHPQQAFCNADWIRAKAVSEKE [SEQ ID NO:

8].

A method as claimed in any preceding claim, wherein the HsTIMP2 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof.

A method as claimed in any preceding claim, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 9 or a sequence of at least 64% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 10.

7. A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more

recombinant proteins and NbPR4 polypeptide in the apoplast of the plant or plant part.

8. A method as claimed in claim 7, wherein the NbPR4 polypeptide comprises a

polypeptide sequence of SEQ ID NO: 16 or a sequence of at least 87% identity thereto or a functional fragment thereof.

9. A method as claimed in claim 7 or claim 8, wherein the NbPR4 polypeptide

comprises a signal peptide capable of directing NbPR4 polypeptide accumulation in the apoplast of a plant.

10. A method as claimed in claim 9, wherein the NbPR4 polypeptide comprises a

polypeptide sequence of SEQ ID NO: 26.

1 1. A method as claimed in any of claims 7 to 10, wherein the NbPR4 polypeptide

comprises; a. the amino acid motif TVRIVDQC [SEQ ID NO: 1 1]; and/or b. the amino acid motif NGGLDLD [SEQ ID NO: 12]; and/or c. the amino acid motif CGRCLRVTNT [SEQ ID NO: 13]; and/or d. the amino acid motif LDTNGVGYQQGHLIVNYEFINCDD [SEQ ID NO: 14]; and/or e. the amino acid motif AGGQSATN VRSTYH LYN PQN I NWDL [SEQ ID NO: 15].

12. A method as claimed in any of claims 7 to 1 1 , wherein the NbPR4 polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof.

13. A method as claimed in any of claims 7 to 12, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 17 or a sequence of at least 72% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 18.

14. A method of increasing the level of one or more recombinant proteins in a plant or plant part, wherein the method comprises co-expressing the one or more recombinant proteins and NbPotI polypeptide in the apoplast of the plant or plant part.

15. A method as claimed in claim 14, wherein the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 23 or a sequence of at least 50% identity thereto or a functional fragment thereof.

16. A method as claimed in claim 14 or claim 15, wherein the NbPotI polypeptide comprises a signal peptide capable of directing NbPotI polypeptide accumulation in the apoplast of a plant.

17. A method as claimed in claim 16, wherein the NbPotI polypeptide comprises a polypeptide sequence of SEQ ID NO: 27.

18. A method as claimed in any of claims 14 to wherein the NbPotI polypeptide comprises; a. the amino acid motif KX1X2WPELVG [SEQ ID NO: 19]; wherein Xi is one of Histidine, Serine, Aspartic Acid, Threonine, Lysine, Glutamine or Valine and X2 is one of Leucine, Serine, Lysine, Glutamic Acid, Glutamine, or Methionine; and/or b. the amino acid motif KHLWPELVG [SEQ ID NO: 20]; and/or c. the amino acid motif NWEFIVIITPTVG [SEQ ID NO: 21]; and/or d. the amino acid motif GMPGKTAKEIIEKENPLV [SEQ ID NO: 22].

19. A method as claimed in any of claims 14 to 18, wherein the NbPotI polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof.

20. A method as claimed in any of claims 14 to 19, wherein the method comprises transforming a plant or plant part with a polynucleotide comprising a polynucleotide sequence of; a. SEQ ID NO: 24 or a sequence of at least 60% identity thereto or a functional fragment thereof; or b. SEQ ID NO: 25.

21. A method as claimed in any preceding claim, wherein the method further comprises increasing the level or expression of NbPR4 polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

22. A method as claimed in any preceding claim, wherein the method further comprises increasing the level or expression of NbPotI polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

23. A method as claimed in any of claims 1 to 20, further comprising increasing the level or expression of NbPR4 polypeptide and of NbPotI polypeptide in the apoplast of the plant or plant part compared with that of an unmodified plant or plant part.

24. A method as claimed in any of claims 6, 13, 20, 21 , 22 or 23, wherein said plant or plant part is transformed with a multiplicity of said polynucleotides; optionally wherein the plant or plant part is transiently transformed with one or more of said polynucleotides, or wherein the plant or plant part has one or more of said

polynucleotides stably incorporated into its genome.

25. A method as claimed in any of claims 6, 13, 20, 21 , 22, 23 or 24, wherein one or more of the polynucleotides is expressed in the plant or plant part and its expression results an increase in the expression or level of the corresponding polypeptide compared to that of an equivalent untransformed plant or plant part.

26. A method as claimed in any preceding claim, wherein the plant or plant part has an increased level of recombinant protein compared to an equivalent plant or plant part, which is modified to express the recombinant protein but lacks expression of HsTIMP2, NbPR4 and NbPotl in the apoplast of the plant or plant part.

27. A method as claimed in any preceding claim, wherein the one or more recombinant proteins is VCR01 and/or Erythropoietin (EPO) and/or Galactose-alpha- 1 ,3-galactose (a-Gal).

28. A modified plant or plant part genetically engineered to be capable of expressing Tissue Inhibitor of Metalloproteinase 2 (HsTIMP2) polypeptide and/or NBPR4 polypeptide and/or Nbpat 1 polypeptide in the apoplast of the plant or plant part.

29. A modified plant or plant part as claimed in claim 28, wherein the HsTIMP2 is as set forth in any of claims 2 to 6: wherein the NBPR4 polypeptide is as set forth in any of the claims 8 to 13; and of claims 8 to 13; and wherein the Nbpati polypeptide is as set forth in any of claims 15 to 19.

30. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 1 or a sequence of at least 63% identity thereto or a functional fragment thereof.

31. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 26 or a sequence of at least 63% identity thereto or a functional fragment thereof.

32. A protease inhibitor comprising the polypeptide sequence of SEQ ID NO: 27 or a sequence of at least 50% identity thereto or a functional fragment thereof.