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HUMANIZATION OF THE N-GLYCOSYLATION PATHWAY IN PLANTS AND PLANT PRODUCTION SYSTEMS Baccalaureate Thesis written by MICHAEL KARBIENER 0240343 in April & May 2005 Supervisor: Ao. Univ. Prof. DI. Dr. LUKAS MACH Department of Applied Plant Sciences and Plant Biotechnology Institute of Applied Genetics and Cell Biology University of Natural Resources and Applied Life Sciences, Vienna BACCALAUREATE THESIS April & May 2005 LIST OF CONTENTS page I. INTRODUCTION 3 II. BASIC MOLECULAR AND CELLULAR BIOLOGY II.1 DNA – Storage of genetic information 4 II.2 From DNA to RNA – Transcription 5 II.3 From RNA to protein – Translation 6 III. POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS III.1 Proteolytic Events 9 III.2 Attachment of Fatty Acids 10 III.3 Attachment of Ions 10 III.4 Glycosylation 10 III.5 Other PTMs 11 IV. DIFFERENCES BETWEEN HUMAN AND PLANT N-GLYCOSYLATION PATHWAY 12 V. STRATEGIES FOR THE HUMANIZATION OF THE PLANT N-GLYCOSYLATION PATHWAY V.1 Inhibition of Glycosylation Strategy 14 V.2 ER Retention Strategy 14 V.3 Inactivation & Implementation Strategies V.3.1 Inactivation of plant Glycosyltransferases 16 V.3.2 Implementation of mammalian Glycosyltransferases 17 V.4 Future Prospects 18 VI. PLANT PRODUCTION SYSTEMS VI.1 General Advantages and Bottlenecks 20 VI.2 Choice of Production Species VI.2.1 Leafy Crops 24 VI.2.2 Cereal and Legume Seeds 25 VI.2.3 Fruit and vegetable Crops 26 VI.2.4 Plant-cell-suspension Cultures 27 VI.2.5 Chloroplast transgenic System 27 VII. CONCLUDING REMARKS 29 ABBREVATIONS 31 REFERENCES 32 MICHAEL KARBIENER 0240343 2 BACCALAUREATE THESIS April & May 2005 I. INTRODUCTION As the knowledge of genes and their regulation in organisms is continually growing, also the construction of more and more sophisticated biochemical production systems proceeds. In the past decades, especially the production of complex human pharmaceuticals was carried out by processes involving only mammalian cells. But genetic engineering has reached a point at which several other cell types can be seriously considered to compete with the animal cell production system: Many researchers have focused their work on the modification of the cells of insects, yeasts, fungi – and plants. The use of plants by men dates back to the stone age. First our ancestors just collected seeds, beers etc. for nourishment, but soon they learned to cultivate the plants, which means the beginning of a process that has led to practically all useful plants today. It was already at that time that people had begun to select or favour certain sets of genes and by this regarding other sets – without being conscious of the present knowledge of molecular biology at all, but already understanding and using the principles of the process called inheritance. Also the role of plants in treatment of diseases has a long history dating back to ancient cultures. Today, millions of years later, we are about to open a new stage in the cultivation of plants, in which they can serve people as valuable producers of therapeutic proteins that maybe soon won´t be able to be distinguished from originally human proteins any more. The major difference between proteins of different eucaryotic species lies in their different variations of post-translational modifications (PTMs). Especially the glycosylation is an important factor for the immunogenicity, the activity and the half life of a pharmaceutical protein derived by an insect cell, a yeast cell, a fungus or a plant. It is the main aim of this essay to summarize the current status of genetic engineering in the N-glycosylation pathway of plants and the remaining differences to the human N-glycosylation pathway, thereby pointing out the problems which will have to be solved to humanize plant-derived proteins completely. Furthermore, the possible variants of plant production systems with their particular advantages and disadvantages shall be described. MICHAEL KARBIENER 0240343 3 BACCALAUREATE THESIS April & May 2005 II. BASIC MOLECULAR AND CELLULAR BIOLOGY This chapter gives a brief overview of the intracellular pathway which leads to gene expression in eucaryotes, as it is first important to know the fundamental process – which is common to all eucaryotes – before enlighting its slight, but crucial differences in different organisms, which will be discussed in the following chapters. Readers familiar with the basics of molecular biology may simply skip this section. Fig. 1: Architecture of an animal cell. This picture may serve as an overview in order to visualize where certain reactions happen and where particular transport routes proceed. II.1 DNA – STORAGE OF GENETIC INFORMATION From a molecular biological point of view, every organism consists of a certain set of genes (e.g. approximately 30.000 in humans). These genes are stored in the nucleus of every cell in the form of DNA, a long polymer built up by monomers called nucleotides. Each nucleotide consists of a C-5-sugar (2`-deoxyribose) and a phosphate by which it is linked to the next nucleotide via a phospho-diester bond, as well as of a base which can be Adenine, Guanine, Cytosine or Thymidine. The MICHAEL KARBIENER 0240343 4 BACCALAUREATE THESIS April & May 2005 characteristic structure of DNA (=the DNA double helix) is made up by two nucleotide polymer chains which align along each other via H-bonds that occur between the nucleotide bases of the two strains. Due to the fact that A can only base-pair with T and C only with G, the two strains of a DNA molecule relate to each other like positive and negative of a picture, containing the same information which lies in the sequence of the four possible nucleotide bases. A gene now can be seen as a certain nucleotide sequence consisting of parts that later will be “translated” into the amino acid sequence of a protein (exons, cf. 2.3), of parts which do not encode amino acids (introns, lying between exons), as well as of so-called regulatory regions (mostly before or after the exon-intron-region). The latter are very important in the cell because they determine whether the information is just storaged without making use of it or whether it is allowed to be “expressed”, which means to be (transcribed and) translated and thus being enabled to carry out its certain function within the cell. This so-called gene regulation is very sophisticated especially in multicellular organisms like humans- due to the fact that only because of such “expression-controlling mechanisms”, different cell types with different size, form and function can exist. The DNA in practically every cell of the 1016 cells of a human (except the germ cells) contains the full hereditary information- but in every particular cell type, only a certain set of genes is expressed throughout the lifetime of this cell. II.2 FROM DNA TO RNA – TRANSCRIPTION As mentioned earlier, the DNA – containing information for all different proteins a cell can synthesize – is stored in the nucleus. However, protein synthesis takes place in the cytoplasm, which means that the information somehow has to be transported there. A second type of nucleotide polymeric molecule called RNA (Ribonucleic acid) serves this function. The molecular difference between DNA and RNA lies in the sugar (being a Ribose in RNA) and in the exchange of the T base for a Uracil base (U) in RNA (which, as T, can only base-pair with A and thus has the same function in the genetic code). In eucaryotes, several different RNAs, all having distinct functions in the cell, are known. The transport of protein information is carried out by the messenger RNA type. Synthesis of this molecule takes place in the nucleus and involves several MICHAEL KARBIENER 0240343 5 BACCALAUREATE THESIS April & May 2005 proteins called general transcription factors, among which the RNA polymerase II is the most important. This enzyme is able to recognize a certain DNA sequence called promoter, binds to it and unwinds the DNA molecule in this region- which means that the base-pairing between the two antiparallel DNA strains is lost. Because of this, A-, C-, G- and U-ribonucleotides (monomers!) can now base pair with the DNA template. The RNA polymerase is able to carry out the polymerisation reaction by connecting single nucleotides (aligned along the DNA strand) via a phosphodiester bond. This polymerisation is continued until the newly synthesized RNA molecule forms a structure called hairpin loop, which causes the RNA polymerase and other associated proteins to stop the transcription process and leave the DNA strand. In this way, each single gene is transcribed, which means that the information has been transformed from the storage form (=DNA-sequence) into the transport form (=RNA-sequence). Before the export of a mRNA, several co- & post-transcriptional modifications take place. Only the most important will be explained now. To the 5’-end, a certain “cap” stucture, and to the 3’-end, a poly-A-tail are added (by specific enzymes), both important as export signals (and thus ensuring that only “mature” mRNA is exported) and as recognition signals at the ribosomes, where translation later will take place. Furthermore, in the process of RNA splicing (carried out by protein-RNA-complexes called spliceosomes) introns are cut out of the gene sequence, giving a mRNA consisting of a continous sequence of amino-acids-encoding nucleotides. Finally, the “ready-to-translate” mRNA molecule is transported to the cytosol through a nuclear pore complex (located in the nuclear membrane). II.3 FROM RNA TO PROTEIN – TRANSLATION Proteins are a second class of cellular polymeric molecules, consisting of 20 different amino acids which are linked by peptide bonds. As with DNA, it is the sequence of the monomers and the length (= the number of connected monomers) that creates the great variety of different proteins. The information lying in the nucleotide sequence of a mRNA molecule must be translated into the amino acid sequence of a protein, a process thus called translation, which is carried out by ribosomes. These are large complexes MICHAEL KARBIENER 0240343 6 BACCALAUREATE THESIS April & May 2005 consisting of 2 protein subunits associated with catalytic rRNA (=ribosomal RNA) molecules. Regulative sequences at the beginning of the mRNA interact with the rRNA via base-pairing, which positions the mRNA in the right way for starting translation. Now, a further class of RNA, the tRNA (=transfer RNA), is involved: At one end, the tRNA has an amino acid covalently bound; at an other region, it contains a specific nucleotide triplet, the anticodon. Different tRNAs can be distinguished according to which amino acid they have bound, as well as to the sequence of their triplets, but the pairing of a certain amino acid to a certain tRNA with a certain triplet is strictly consistent, which is crucial for the correct translation of genetic information. The ribosome has a site where a tRNA can get access to the codon, which is a nucleotide triplet of the mRNA. Only the right matching between codon and anticodon (via base-pairs) stabilizes a certain tRNA long enough that its amino acid is linked to a growing polypeptide chain (an endothermic reaction for which GTP hydrolysis is necessary). Moving of the ribosome on the mRNA template enables the next cycle of tRNA-mRNA-base-pairing and amino acid transfer, and so translation proceeds stepby-step. Looking back on the pathway of information, it is now clear the exons of the DNA sequence of a gene encode the amino acid sequence of a protein in a way that always three consecutive nucleotides encode one amino acid. Simple combinatorical thoughts show that 4 x 4 x 4 = 64 different base triplets are possible. Because there do exist only 20 different amino acids in proteins, the so-called genetic code is degenerated, which means that some amino acids are encoded by more than just one triplet. (As a consequence, there do exist several tRNAs with the same amino acid bound, but a different anticodon.) At the end of its coding sequence, each mRNA molecule contains a triplet that does not encode an amino acid. Three of the 64 possible triplets are such termination signals, which prevent further elongation of the polypeptide chain (as no fitting tRNA for them exists) and lead to the dissociation of the ribosome-mRNA-Polypeptidecomplex, thereby ending each particular translation event. For a protein, according to its final destination, two fundamentally different possibilities exist: It can either be synthesized in the cytosol completely or it can be directed to the secretory pathway. The first way is the fate of all cytosolic, MICHAEL KARBIENER 0240343 7 BACCALAUREATE THESIS April & May 2005 mitochondrial, chloroplast, peroxisomal and nuclear proteins, and the transport to the specific cell site (if not the cytoplasm) occurs post-translational. The second way involves a co-translational transport mechanism: Every protein destined for the secretory pathway has a specific N-terminal signal sequence which, when synthesized at the beginning of translation, interacts with a cytosolic protein named signal-recognition particle (SRP). Translation is stopped and the ribosome-mRNA-Peptide-SRP-complex is directed to the ER, where interactions with the SRP and its receptor in the ER membrane facilitate the import of the N-terminal end of the nascent protein. Only then translation can proceed (with the ribosomes tightly bound to the surface of the ER), and the protein can either be inserted in the ER lumen completely or anchored in the ER membrane (depending on further signal sequences). All proteins of the cytoplasmic, ER, Golgi, endosomal and lysosomal membranes as well as non-membrane proteins in the lumen of ER, Golgi, endosomes and lysosomes and proteins whose final destination is the extracellular space migrate through the secretory pathway. It is also in this pathway where several proteins undergo most post-translational modifications, as will be discussed the next chapter. Fig. 2: From gene to protein, secretory pathway. This picture summarizes the way in which the information of a certain gene is transformed into a mature protein: First, transcription, RNA splicing and transport to the cytosol take place. In this case, the gene contains a N-terminal signal sequence, which after initiation of translation leads to co-translational insertion of the polypeptide chain into the ER lumen. The protein is then transported through the secretory pathway, where most PTMs occur. MICHAEL KARBIENER 0240343 8 BACCALAUREATE THESIS April & May 2005 III. POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS The secretory pathway starts with a protein´s direction to the ER. From there, lumenal as well as membrane proteins are passed on to the Golgi network by vesicular transport. During this migration, a protein can undergo different PTMs, carried out by enzymes exclusively located at certain sites of this pathway. The vast majority of therapeutic proteins undergo several PTMs, which are required for their bioactivity, pharmacokinetics, stability and solubility [1]. While the steps from DNA to protein mentioned in chapter 2 are highly similar in all eucaryotic organisms, from now on protein processing can – but not necessarily needs to – vary in different species like humans and plants. This chapter depicts an overview of the different kinds of PTMs which are generally possible in higher eucaryotes. N-glycosylation is only mentioned shortly at this point to complete the list of PTMs, being discussed separately in an own chapter afterwards. III.1 PROTEOLYTIC EVENTS Proteolysis is the degradation of a protein by hydrolysis of one or more of its peptide bonds [2]. In mammalian cells, many secreted proteins are synthesized as preproteins, and are further matured co- and posttranslationally by soluble or membrane-bound proteinases during their transport through the secretory pathway [1]. One prominent example is the cleavage of the N-terminal signal sequence by a signal peptidase located in the ER membrane (with its catalytic domain on the lumenal side). Also propeptides, which have a regulating function concerning the activity of a certain protein and its interaction with other proteins, are lost during the process of “proteolytic maturation”. One example is procollagen, the precursor of mature collagen, where the cleavage of the propeptides occurs not before the secretion of the molecule to the extracellular matrix. Only then interactions between molecules can happen to give higher order structures (collagen fibrils). Procollagen is also one of several propeptides for which, when engineered into a plant, correct processing (resulting in biologically active forms) has been shown [1]. MICHAEL KARBIENER 0240343 9 BACCALAUREATE THESIS April & May 2005 III.2 ATTACHMENT OF FATTY ACIDS Covalent linkage of a protein to a fatty acid provides a possibility for retaining the protein closely at a membrane. A well studied example is the Glycosylphosphatidylinositol(GPI)-anchor, in which the C-terminal Asp of certain proteins is connected (by a phosphoethanolamine and an oligosaccharide unit) to a Glycerolphosphate unit. Also the N-terminal addition of a fatty acid structure (without linking sugar chains) is possible (e.g. N-myristoylation). III.3 ATTACHMENT OF IONS A) PHOSPHORYLATION In both plants and humans, phosphate groups occur added to Ser, Thr and Tyr, more seldom to other kinds of amino acid side chains [1]. B) SULFATION In humans, sulfate groups are added to Tyr residues of proteins or to the N-glycan; this PTM has not been discovered yet in the secretory pathway of plants [1]. C) GAMMA-CARBOXYLATION In the human ER, glutamate residues of proteins can be transformed into γ– carboxyglutamate, a reaction which is vitamin-K-dependent. No comparable modification has been discovered in plants so far. III.4 GLYCOSYLATION The process in which sugar monomers or oligomers are added enzymatically to certain amino acid residues of a protein is termed glycosylation. The following types of glycosylation can be distinguished: A) N-GLYCOSYLATION A precursor oligosaccharide (Glc3Man9GlcNAc2) is added to the amide nitrogen of an Asn amino acid [3]. Only Asn residues of the consensus sequence Asn-XSer/Thr – where X can be any amino acid except proline– can be glycosylated (but, most probably to conformational factors, in a protein not each Asn that is part of a consensus sequence is glycosylated) [4]. MICHAEL KARBIENER 0240343 10 BACCALAUREATE THESIS April & May 2005 B) O-GLYCOSYLATION Generally, to all amino acids containing an OH-group – Ser, Thr, Tyr, hydroxyproline and hydroxylysine –, a sugar can be added covalently to give an O-glycosylated glycoprotein. So far, only little attention has been paid to the O-glycosylation status of therapeutic proteins produced in transgenic plants, although strong differences in the Oglycosylation machinery compared to humans exist [1]. III.5 OTHER PTMS A) HYDROXYLATION Hydroxyl-residues can be found added to Pro and Lys in humans as well as in plants, although the involved enzymes of both species have different recognition sequences. B) ACETYLATION Acetylation of proteins, either on various amino-terminal residues or on the epsilonamino group of Lys residues, is catalyzed by a wide range of acetyltransferases. Acetylation of the amino-residue may confer additional stability on these proteins [1], but the function in plants has not been understood yet. An expression system for a therapeutic protein is usually chosen considering glycosylation requirements, due to the fact that the attachment of carbohydrates to the polypeptide backbone strongly affects the physico-chemical properties of a protein, including resistance to thermal denaturation, protection from proteolytic degradation and solubility [3]. Moreover, also essential biological functions such as immunogenicity, clearance rate, specific activity and ligand-receptor interaction are influenced by glycosylation. Altogether, these factors make it necessary to explore the differences of plant and human glycosylation events in detail in order to get to know at which points the plant glycosylation pathway has to be changed for production of completely humanized proteins (see Chapter IV). MICHAEL KARBIENER 0240343 11 BACCALAUREATE THESIS April & May 2005 IV. DIFFERENCES BETWEEN HUMAN AND PLANT N-GLYCOSYLATION PATHWAYS In plant cells, like in other eucaryotic cells, N-glycosylation starts in the ER lumen by the co-translational transfer of an oligosaccharide precursor, Glc3Man9GlcNAc2, from a Dolichol (which anchors the oligsaccharide at the ER membrane) onto specific Asn residues constitutive of the N-glycosylation sequences, Asn-X-Ser/Thr. This step is done by the Oligosaccharyltransferase complex, a large enzymatic complex located in the ER membrane. Once transferred onto the nascent protein and while the glycoprotein is transported along the secretory pathway, the oligosaccharide N-linked to Asn (N-glycan) undergoes several maturation steps involving the removal and the addition of sugar residues in the ER and the Golgi complex [3]. Plant and mammalian N-glycan maturations differ only in the late Golgi apparatus (see Fig. 3): • Core α(1,6)-Fuc residues and terminal NeuAc are formed in mammals, whereas • Bisecting β(1,2)-Xyl and core α(1,3)-Fuc residues are formed in plants [1]. This means that, if a mammalian glycoprotein is expressed in plants, it will have a different glycan structure caused by the differences in plant and human N-glycan processing. Apart from the principal possibility that the function of an incorrectly glycosylated protein can be lost or altered (e.g. due to misfolding), also another problem can arise when using a plant-made protein for therapy: core Xyl and core α(1,3) Fuc epitopes are known to be important IgE binding carbohydrate determinants of plant allergens [5]. Furthermore, it has been shown that the immunization of goats or rabbits with plant glycoproteins elicits the production of core Xyl- and core α(1,3)Fuc-specific antibodies, which raises the question of immunogenicity in humans: Is a certain N-glycosylated, plant-made protein able to cause a response from the human adaptive immune system because of its different glycan structure? –This MICHAEL KARBIENER 0240343 12 BACCALAUREATE THESIS April & May 2005 question still remains a pending issue. In the context of human therapy, elicitation of immune responses in humans by specific plant glyco-antigens could be a major concern if people have prolonged exposure to large quantities of plant-derived glycoproteins, as may be required for certain in vivo treatments [5]. For example, when high amounts of plant-made antibodies will be repetitively injected for cancer therapy in humans, the presence of immunogenic N-glycans on these proteins could at least induce their fast clearance from the blood stream mediated by carbohydratespecific antibodies [3]. Fig. 3: Addition and processing of N-linked glycans in the ER and Golgi apparatus in plants and mammals. A precursor oligosaccharide assembled onto a lipid carrier is transferred on specific Asn residues of the nascent growing polypeptide. The N-glycan is then trimmed with the removal of glucosyl and most mannosyl residues. Differences in the processing of plant and mammalian complex N-glycans occur during late Golgi maturation events. It is obvious that the tools of today´s molecular biology can be used for trying to overcome these problems by humanization of the plant N-glycosylation pathway. Genetic engineering provides a chance for fully using the exciting potential of the plant production system, as the next chapter will discuss. MICHAEL KARBIENER 0240343 13 BACCALAUREATE THESIS April & May 2005 V. STRATEGIES FOR THE HUMANIZATION OF THE PLANT N-GLYCOSYLATION PATHWAY V.1 INHIBITION OF GLYCOSYLATION STRATEGY One of the most drastic approaches for the humanization of plant-made glycoproteins is to prevent the addition of immunogenic N-glycans to the polypeptide. This can easily be acquired by mutating the nucleotide (and thereby the amino acid) sequence of the final product in a way that a potential N-glycosylation signal sequence (Asn –X – Ser/Thr) is lost. For an antibody, this strategy mostly will not inactivate its antigen binding function. However, many pharmaceuticals, including antibodies for their effector functions, such as the triggering of the immune response, require glycosylation for an increased in vivo activity and stability [6]. Additionally, it has been discovered that both the halflife and biological activity of a recombinant protein can be increased by the addition of N-glycans. This further illustrates a current tendency in glyco-engineering to increase, and not to reduce, the number of glycosylation sites on recombinant pharmaceuticals [6]. Finally, by avoiding glycosylation processes at all, one must admit that, for this strategy (as well as for the next), the term “humanization” must be put in perspective, as not a human-like glycan structure, but just an avoidance of human immune responses is archieved. V.2 ER RETENTION STRATEGY As already mentioned, the journey of proteins along the secretory pathway makes use of vesicular transport mechanisms. This means that such a lumenal or membrane cargo protein (=a protein that shall be transported to the next station of the pathway, e.g. from the ER to the cis-Golgi) selectively binds to certain cargo receptors which are transmembrane proteins that themselves interact with cytosolic coat proteins. The latter form the outside of so-called coated vesicles, which bud off from a certain compartment containing a collection of selected cargo proteins MICHAEL KARBIENER 0240343 14 BACCALAUREATE THESIS April & May 2005 destined for another compartment which they enter by fusion of (at that time already “uncoated”) vesicle and target membrane. As a result of the specific interactions between different cargo proteins with their different cargo receptors, the ER and especially the Golgi apparatus can serve their function as ”sorting station”. But this mechanism can only work properly if a retrieval flux of cargo receptors (together with membrane components) to the compartment from which they have budded off is maintained. For the ER, this process depends on ER retrieval signals, which are short amino acid sequences at the extreme Cterminal end of ER resident proteins (= proteins that are destined to carry out their function in the ER and thus must be returned to it if escaped). Soluble ER proteins contain the KDEL (Lys-Asp-Glu-Leu) retrieval sequence that has been discovered because normally secreted proteins, to which this sequence had been C-terminally added, accumulated in the ER [2]. The ER retention strategy makes use of this retrieval pathway by preventing that therapeutic proteins reach the late Golgi, where the plant-specific maturations take place. If to a certain protein of interest, a H/KDEL sequence is added to its Cterminus, the protein is successfully stored in the plant ER and has mainly glycans of the high-mannose-type, which are oligosaccharide structures that mammals and plants have in common and therefore are probably not immunogenic [6]. The efficiency of this strategy has been proved in a recent experiment where the gene for a human antibody (=mAbM) against the rabies virus was introduced and expressed in tobacco plants. The nucleotide sequence of the heavy chain was modified by the addition of the ER retention signal KDEL. In contrast to the human antibody, which contains several complex N-glycans, about 90% of the plant-made antibodies (=mAbP) displayed oligomannose N-glycans [7]. The remaining fraction contained the immunogenic β(1,2)-xyl glyco-epitope [6], which indicates that these antibodies have first been transported down to the medial Golgi – where β(1,2)xylosyltransferase is located – but then were retrieved and transported back to the ER (due to their KDEL sequence). However, no antibody was α(1,3)-fucosylated, a glycan modification occuring in the trans-Golgi site. Compared to the human anti-rabies antibody, the plant-derived counterpart showed the same neutralizing and protective efficacy which can easily be imagined as the different glycosylation pattern – occuring on the CH2 domain – does not influence the affinity of the VH and VL domains for the antigen. But because of its high-mannose MICHAEL KARBIENER 0240343 15 BACCALAUREATE THESIS April & May 2005 N-glycan structure, the half-life of mAbP was much shorter than that of the mAbM, which can be explained by the binding of the antibodies to Man receptors such as the macrophage Man receptor in the liver [7]. V.3 INACTIVATION & IMPLEMENTATION STRATEGIES Although directioning more towards humanization than the “Inhibition of Glycosylation Strategy”, also the “ER Retention Strategy” isn´t a tool for generating human-identical glycan structures and thus in many cases only a suboptimal solution. The remaining possible strategies which will be discussed now depict the most ambitious, but also the most promising possibilities of humanizing plant-made glycoproteins. V.3.1 INACTIVATION OF PLANT GLYCOSYLTRANSFERASES In a plant cell, the main target enzymes to be inactivated are the β(1,2)xylosyltransferase and the α(1,3)-fucosyltransferase, which are responsible for the plant-specific transfer of Xyl and Fuc onto the growing N-glycan (cf. Fig. 4). The first experiments have been carried out with the moss Physcomitrella patens, as this is the only plant system showing a high frequency of homologous recombination [6], which makes the knockout of certain genes possible in a relatively easy way. Furthermore, in P. patens, N-glycosylation is highly similar to that in higher plants as Arabidopsis thaliana, and so these first results can pave the way to inactivation strategies in those plants that are interesting production systems for pharmaceutical proteins. Knocking out the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes successfully permitted the disappearance of the plant-specific glyco epitopes without any effect on protein secretion in the moss [6]. Apart from this, analysis of these plant enzymes has presented another valuable information: Plant glycosyltransferases, being type II membrane proteins as their human relatives, contain amino acid sequences responsible for their location at certain points in the secretory pathway. A detailed characterization of β(1,2)-xylosyltransferase from A. thaliana has revealed that the first 36 amino acids (cytosolic tail and transmembrane domain) are responsible for its Golgi retention, and furthermore contain information for sub-Golgi MICHAEL KARBIENER 0240343 16 BACCALAUREATE THESIS April & May 2005 compartment targeting [6]. By the analysis of several other glycosyltransferases, a panel of specific signals that are sufficient for protein targeting to the different Golgi subcompartments is currently provided- which will help to target exogenous glycosyltransferases to the plant Golgi apparatus for an optimal efficiency in the engineering of the glycosylation pathway [6]. Fig. 4: Differences in the structure of complex N-glycans between plants and humans. To humanize the recombinant proteins that are made in plants, α(1,3)Fuc and β(1,2)-Xyl residues (yellow) must be removed, whereas core Fuc, Gal and NeuAc residues (red) must be added. Blue residues are common to plants and humans. V.3.2 IMPLEMENTATION OF MAMMALIAN GLYCOSYLTRANSFERASES As natural plant-derived N-glycans do not only contain sugars that are absent in their mammalian relatives, but also lack some carbohydrate structures present in humans, the expression of mammalian glycosyltransferases in plants is a promising technique for humanization. In first experiments, human β(1,4)-galactosyltransferase was introduced into the genome of tobacco plants. In these modified organisms, 30% of N-glycans carried by an antibody (= a human model protein) beared the terminal N-acetyllactosamine sequences of the mammalian type (cf. Fig 4). The efficiency of this strategy was further increased when a targeted expression of the enzyme was obtained after fusion of the catalytic domain of this glycosyltransferase with the first 54 amino acids of A. thaliana β(1,2)-xylosyltransferase [6]. Nevertheless, tobacco plants probably won´t be the ideal production system for pharmaceutial glycoproteins, MICHAEL KARBIENER 0240343 as the action of the implemented human 17 BACCALAUREATE THESIS April & May 2005 β(1,4)galactosyltransferase seems to lead to a highly complex mixture of N-glycans (because the human enzymes and the plant´s own glycosyltransferases “fight” for the same substrate), which would extremely complicate pucification processes. At least for antibodies, a better organism seems to have been found in Alfalfa: Expression of a certain monoclonal antibody (C5-1) in this plant resulted in the production of plant-derived IgG1, which is N-glycosylated by a predominant glycan having an α(1,3)-Fuc and a β(1,2)-Xyl attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal linked N-glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having an Nglycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible antibody [8]. V.4 FUTURE PROSPECTS Production of an IgG antibody in transgenic plants was first described in 1989 [9]. Considering the fast development and promising results of the last 15 years, the first production of a PMP with a human-identical N-glycan structure (as “proof of principle”) can easily arrive in the near future. Current efforts are concentrated on the association of implementation strategies with inhibition strategies, which shall give Nglycans containing the human-specific sugar residues (core α(1,6)-Fuc, β(1,4) Gal, terminal NeuAc), but lacking the plant-specific glycoepitopes α(1,3)-Fuc and β(1,2)Xyl. For antibodies, only few steps are missing towards their production with an Nglycosylation profile identical to that observed in mammals: • Still a better inactivation of plant β(1,2)-xylosyltransferase and α(1,3)fucosyltransferase must be archieved and • the performance of implemented human β(1,4)-galactosyltransferase must be improved, which could be enabled by a better control of its targeting in the Golgi cisternae. Compared with other plasma proteins, antibodies present a rather simple Nglycosylation, with partial terminal β(1,4)-galactosylation and very few terminal sialic acids. However, sialic acids are very important for the clearance of many mammalian MICHAEL KARBIENER 0240343 18 BACCALAUREATE THESIS April & May 2005 plasma proteins of pharmaceutical interest [6]. If there are no NeuAc residues present on them, they will be eliminated quickly from the bloodstream due to interactions with galactose-specific receptors on the surface of hepatic cells. The special problem with sialylation in plants is that the presence of NeuAc in plants has not been discovered at all in any species. So, for a terminally sialylated N-glycan, not only the specific glycosyltransferase (=sialyltransferase), but moreover the whole set of enzymes which can manage in vivo biosynthesis of NeuAc and a transporter for its import into the Golgi lumen would have to be expressed in plants. This means the engineering of at least five heterologous genes, which should not only be expressed in a stable manner, but should also be active and correctly targeted in a plant cell [6]. Another possibility would be the production of relevant PMPs bearing Nglycan-precursors, which – after isolation from the plant tissue and purification – could be modified by a sialyltransferase in vitro. In summary it may be said that the production of plantibodies pertains a today´s technology, whereas the production of sialylated PMPs at large remains a much more ambitious objective for plant biotechnology [6]. MICHAEL KARBIENER 0240343 19 BACCALAUREATE THESIS April & May 2005 VI. PLANT PRODUCTION SYSTEMS The success of any biotechnological system used for the production of therapeutic agents depends on three distinct groups of factors: technological factors, economical factors and – last but not least – factors concerning public acceptance. Because most genes can be expressed in many different systems, it is essential to determine which system offers the most advantages for the production of a recombinant protein [10]. This chapter shall give a portrait of plant production systems: First, the general advantages, but also the difficulties concerning PMPs are listed. With regard to this, in the second part, different ways of producing a pharmaceutical in plants shall be discussed, thereby enlighting the possible chances on the market for certain products in more detail. VI.1 GENERAL ADVANTAGES AND BOTTLENECKS Today, many therapeutic agents in principle could be employed to successfully fight many serious diseases- if they were only affordable to be produced on large industrial scale. For example, approximately 30 million children are born each year that are not adequately immunized by modern standards. WHO and various other organizations have stressed the need for new technology to create additional vaccines against infectious diseases to increase global immunization compliance and to decrease costs of delivery [9]. Facts like these immediately rise the idea of using transgenic plants for molecular farming, having several advantages compared with other production systems. First, plant systems are more economical than industrial facilities using fermentation or bioreactor systems [10]. It is estimated that recombinant proteins can be produced in plants at 2-10% of the cost of microbial fermentation systems and at 0,1% of the cost of mammalian cell cultures (cf. Fig. 5), although this depends on the product yield [11]. MICHAEL KARBIENER 0240343 20 BACCALAUREATE THESIS April & May 2005 Fig. 5: Costs per gram for purified immunoglobulin A produced by different expression systems. Cost for mammalian cell culture are derived from industry costs for cell culture and purification facilities. Costs for transgenic goats are derived from publicly available estimates from Genzyme Transgenics (Farmingham, MA, USA). Costs for plants compare green biomass (120.00 tonne ha-1) and seed production (7.5 tonne ha-1). Cost differences are based primarily on production costs, and it was assumed that purification costs and losses during purification will be the same for all systems. Second, the technology is already available for harvesting and processing plants and plant products on a large scale [10]. In this respect, fermentation systems and transgenic animals have only limited potential, whereas the scale of plant-based production can be modulated rapidly in response to market demand simply by using more or less land as required [11]. Third, plants are considered a much safer system than both microbes and animals because they generally lack human pathogens, oncogenic DNA sequences and endotoxins [11]. This last general advantage also builds the bridge to the other side of the coin, as several environmental concerns have been raised by interest groups to confuse public perception. One certainly cannot deny that there do exist biosafety risks, among which the most important are: MICHAEL KARBIENER 0240343 21 BACCALAUREATE THESIS • April & May 2005 transgene spread by pollen dispersal, seed dispersal and horizontal gene transfer, • the effects of potentially toxic recombinant proteins on herbivores, pollinating insects and microorganisms in the rhizosphere and • the possibility that genetically modified plant material could enter the food chain [12]. But it is also a fact that there exist several strategies for each problem in order to minimize the particular risks: • Transgene spread by pollen dispersal can be adressed by several physical and genetic barrier techniques, as well as by the choice of a suitable production crop that does not outcross with wild plants near the production site [12]. • The possible negative effect of recombinant proteins on non-target organisms can be avoided by the use of regulated promotors to restrict transgene expression to particular organs (e.g. seeds), or to induce protein expression at particular times [12]. • Genetically modified plant production organisms could carry phenotypic modifications, e.g. pigmentation (white tomatoes,...), which would made them easily distinguishable from their natural counterparts. Many other levels of safety can be built into systems, such as geographical isolation, differential planting seasons, the use of male-sterile plants and transplastomic plants (cf. 6.2.5) and the expression of recombinant proteins as inactive precursors which would have to be proteolytically cleaved before showing biological activity [12]. Nevertheless, governmental approval and regulation of PMPs are even more stringent than for pharmaceuticals produced with traditional technology, at least in the US and other industrialized countries [9]. So, the progress of plant-derived therapeutic agents through preclinical development and clinical trials presents a significant bottleneck [11]. At the end of 2003, only two PMPs – human gastric lipase and a recombinant antibody used for the prevention of dental caries – were being tested in phase II clinical trials [11], thereby representing the two plant-made substances being nearest to an entrance of the market. Increasing the yields of desired recombinant proteins represents a further bottleneck. Apart from the advantages of certain production species (highlighted at MICHAEL KARBIENER 0240343 22 BACCALAUREATE THESIS April & May 2005 the respective systems in 6.2), expression-construct design must optimize all stages of gene expression, from transcription to protein stability [12]. General points of optimization are the promoter and the polyadenylation site of a certain gene (often, the 35S transcripts of the cauliflower mosaic virus (CaMV) are used). If it does not reduce the product quality, yields generally can be made twofold to tenfold greater by ER retention (compared to secretion) [12]. Product purification creates another problem, as the production costs of a recombinant protein depend significantly on the required purity because >85% of expenditure reflects downstream processing rather than production per se [11]. This leads back to the challenge of achieving large yields of PMP per unit biomass, as the costs for purification decrease with increasing concentration of the desired product. As with the yield problem, specific advantages of certain production species concerning purification will be mentioned in the following section. Finally, the production timescale must be mentioned as a factor in which many other considerable production systems, especially yeast and bacteria, have the advantage of shorter cycles compared to the rather slow growth of many plants. But several plant-based expression platforms provide possibilities for speeding up production processes: e.g. agroinfiltrated leaves. These are transformed with Agrobacerium tumefaciens, which results in the transient expression of recombinant proteins and thereby is a useful strategy for testing expression constructs and obtaining small amounts of protein for analysis before going to the expense of transgenics [12]. Also virus-infected plants can rapidly set on protein production and enable the establishment of a transgenic plant line within several weeks. Concluding, Table 1 summarizes the different factors that together make up the performance of a certain production system and compares the plant system to the other available alternatives. MICHAEL KARBIENER 0240343 23 BACCALAUREATE THESIS April & May 2005 Table 1: Comparison of production systems for recombinant human pharmaceutical proteins VI.2 CHOICE OF PRODUCTION SPECIES VI.2.1 LEAFY CROPS Amongst leafy crops, Tobacco is a major candidate for the production of recombinant proteins. Tobacco has an established history as a model system for molecular farming and is the most widely used species for the production of recombinant pharmaceutical proteins at the research-laboratory level [12]. The major advantages of tobacco include the well-established technology for gene transfer and expression, high biomass yield, prolific seed production and the existence of a large-scale processing infrastructure [11]. Furthermore, the risk of contamination of food or feed chains is minimized, as tobacco is neither a food nor a feed crop. Although many tobacco cultivars produce high levels of toxic alkaloids, there are low-alkaloid varieties that can be used for the production of pharmaceutic proteins. If a protein of interest is endowed with the fitting signal sequence, it is directed to the secretory pathway of the transgenic tobacco plant and finally secreted to the root or leaf surface. This creates the chance for obtaining the PMP without having to harvest the plant and extract the protein from the cytoplasm. A cultivation of the plants in a hydrophonic system could enable the continuous production of the therapeutic agent which could be purified from the culture medium relatively easily. However, PMPs derived from the tobacco system have the disadvantage of rather heterogenous N-glycan structures (cf. 5.3.2). As a general rule, the higher the homogeneity of the product, the higher the probability that reproducibility will be attained from batch to batch [8]. Without a high level of reproducibility, no production system will ever pass clinical trials. MICHAEL KARBIENER 0240343 24 BACCALAUREATE THESIS April & May 2005 This has led to the investigation of alternative leafy crops such as alfalfa, soybean or lettuce. Apart from its more promising N-glycan trimming (cf. 5.3.2), alfalfa is particularly useful because it has a large dry biomass yield per hectare and can be harvested up to nine times a year [11]. Furthermore, alfalfa and soybean have the advantage of using atmospheric nitrogen through nitrogen fixation, thereby reducing the need for chemical inputs [12]. In general, the productivity of green tissue exceeds the productivity of seeds. Thus, leafy crops are a valuable system for the production of PMPs that are needed in great masses, such as antibodies or vaccines. One of the greatest disadvantages of leafy crops is that recombinant proteins are synthesized in an aqueous environment and are often unstable, resulting in low yields [11]. The leaves must be frozen or dried for transport, or processed soon after harvest to extract useful amounts of the product. In this respect, seeds show their great strength, as the following section will explain. VI.2.2 CEREAL AND LEGUME SEEDS Compared to expression in leaves, any protein expressed in seeds will have a significantly higher half-life. The inside of a seed gives an isolated environment with a very low activity of water, thereby creating the perfect biochemical conditions to promote stable protein accumulation, either as protein bodies or in storage vacuoles, which are derived from the secretory pathway. It has been shown that antibodies that are expressed in seeds remain stable for at least three years at room temperature (!) with no detectable loss of activity [12]. This could create enormous savings in the field of storage and transport costs and enable to satisfy the demand for passive immunization in regions without the appropriate technical facilities, such as in the developing world. The specific expression of recombinant proteins (only!) in the seeds has also biosafety advantages in that it reduces exposure to herbivores and other non-target organisms [11]. However, it is necessary for the transgenic plants to go through a flowering cycle to produce seeds, which means that pollen release happens. On the contrary, proteins produced in vegetative organs can be harvested before flowering, therefore preventing the release of pollen and eliminating gene flow by pollen transfer [11]. Another biosafety concern relates to the potential for genes to spread into crops that are grown for food purposes [12]. MICHAEL KARBIENER 0240343 25 BACCALAUREATE THESIS April & May 2005 So far, rice, wheat and maize (=cereals), and also pea and soybean (=legumes) have been investigated for seed-based production, with rice having showed the highest yields per unit biomass [11]. VI.2.3 FRUIT AND VEGETABLE CROPS “Edible vaccines” is one of the most popular catchwords concerning PMPs. If a certain therapeutic protein is expressed in a fruit or a vegetable, it can be administered straight away – without any expensive purification process – by eating of the raw plant. Apart from human biopharmaceuticals (like human growth hormone or human serum albumin) and antibodies, recombinant vaccines are the third big class of proteins which have been expressed in transgenic plants so far. The WHO and other organizations have recently emphasized the needs for needle-free and heat-stable vaccines as key elements of improved immunization strategies [9]. Both of them could be satisfied, even in an affordable way, by transgenic plants. The first vaccine expressed in plants was the hepatitis B virus (HBV) surface antigen (1992). Since this time, the concept of oral vaccination with raw fruit, vegetables, but also leaves and seeds has risen in popularity [12]. Today, not only the HBV vaccine, but also two further vaccine candidates – the heat-labile toxin B subunit (LTB) of enterotoxigenic E. coli and the capsid protein of the Norwalk virus (NVCP) – have reached the clinical trials stage, all of them produced in potato [12]. Apart from potatoes, which are the major system for vaccine production, bananas are attractive vehicles for edible vaccine distribution because they are widely grown and consumed by both children and adults in Africa where vaccination programmes are badly needed [11]. However, a major concern with oral vaccines is the degradation of protein components in the stomach and gut before they can elicit an immune response [10]. Several delivery vehicles have already been developed to overcome this problem, e.g. liposomes or transgenic plant tissues. An interesting strategy for the “market entry” of plant-derived vaccines is to use them for animal vaccination first [9]. For human vaccines, plant-derived vaccines may first be used as boosting vaccines, after primary immunization with vaccines produced by more traditional techniques. If booster immunization succeeds, the next step would be to try primary immunization [9]. MICHAEL KARBIENER 0240343 26 BACCALAUREATE THESIS April & May 2005 VI.2.4 PLANT-CELL-SUSPENSION CULTURES Continuous agitation of callus tissue results in a homogenous suspension of plant cells which can be cultivated in fermenters such as microbial production systems. Such a production system enables not only sterile, but also defined production conditions, which is not the case for the other systems so far described. Batch, fedbatch, perfusion and continuous fermentation can be realised, each of them used when being the optimal variant for a certain PMP. But by gaining this high level of controllability, one loses the big advantage of low costs which is provided by transgenic plants. Nevertheless, a wide panel of recombinant antibodies has already been expressed in tobacco- and rice-cellsuspension cultures. VI.2.5 CHLOROPLAST TRANSGENIC SYSTEM Generally, levels of pharmaceutical proteins produced in transgenic plants have been less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified [10]. For example, even though Norwalk virus capsid protein expressed in potatoes caused oral immunization when consumed as food, expression levels are too low for large-scale oral administration (0.37% of total soluble protein) [10]. For other human proteins expressed in plants, the concentration was even orders of magnitude smaller (e.g. erythropoietin 0.003%), which highlights the importance of finding solutions for the yield bottleneck. Introducing of foreign DNA into the chloroplast genome of a plant provides an effective stategy for achieving high yields of the desired product. There are several thousand chloroplasts in every photosynthetic cell of a plant, giving a high transgene-copy number. Furthermore, neither gene silencing (which is a common phenonemon with nuclear transformation) nor position effects (the possible extension of heterochromatin areas on chromosomes, which prevents gene expression) have been observed in genetically modified chloroplasts yet. Altogether, these factors lead to astonishing levels of expression, in the best cases exceeding 25% of the total soluble protein [12]. Another advantage of transplastomic plants is that several environmental concerns, e.g. pollen release, lose their relevance. Chloroplasts are also able to fold and MICHAEL KARBIENER 0240343 27 BACCALAUREATE THESIS April & May 2005 assemble oligomeric proteins correctly, which has been proved with the expression of the cholera toxin B subunit [12]. Nevertheless, one disadvantage of the chloroplast transgenic system is that plastids – as they are descendants of procaryotic organisms – do not carry out glycosylation. This makes the system useless for the production of glycoproteins in cases in which the glycan-chain structure is crucial for protein activity [12]. MICHAEL KARBIENER 0240343 28 BACCALAUREATE THESIS April & May 2005 VII. CONCLUDING REMARKS From a philosophical point of view, the importance of plants for mankind has had two different levels so far: First, the production of free oxygen, without which evolution would not have proceeded the way it has. Second, the assimilation of carbon dioxide, giving rise to organic plant tissue, which primarily has provided nourishment for all those organisms that were not able to live on inorganic material themselves, but also useful organic materials like wood – and also a broad panel of substances which were discovered because of their different effects against diseases. At the moment, it seems as a third level is about to arise and join the other two: the production of pharmaceuticals which are originally substances synthesized by humans exclusively. Research over the past 10 years has shown that the expression of even multimeric human proteins in plants can be done in a way that correct folding and assembly is achieved. Differences between plant and human PTMs are the challenges for molecular biologists. N-Glycosylation is one of the most important PTMs, as the carbohydrate structure of a glycoprotein can strongly influence its enzymatic activity, half life and immunogenicity. The aim is that one day plant-derived human glycoproteins won´t be distinguishable from their relatives synthesized in humans any more. This process of humanization on the one hand involves the knockout of plant-specific glycosyltransferases, β(1,2)-xylosyltransferase and α(1,3)-fucosyltransferase. On the other hand, the genes for human glycosyltransferases which are missing in plants must be introduced into the plant genome, successfully expressed and specifically positioned at exactly those points of the secretory pathway where they can carry out their N-glycan modifications in a way they originally do in their natural environment (humans). The implementation of human β(1,4)-galactosyltransferase has already been done and its action in plants merely remains to be optimized. As in plants NeuAc has not been discovered at all so far, it is a much more difficult task to introduce all the genes which would be needed for terminally sialylated PMPs. This big aim will still take years of research to be realised. The big differcence to all other production systems for pharmaceuticals (e.g. mammalian cells, bacteria, yeasts, fungi) is the big potential of plant systems: Production of a PMP in leaves, seeds or fruits can provide a relatively cheap, safe MICHAEL KARBIENER 0240343 29 BACCALAUREATE THESIS April & May 2005 and “easy-to-scale-up” technology. From an economical point of view, plants might one day surpass the other production systems, if the yields of desired recombinant protein can be further increased. But probably commercial success of the plant production system is not as much determined by solving the technological hurdles as it is by biosafety issues and the development of public acceptance. Only if all of these problems can be overcome, it might be possible one day that therapeutic agents can be produced on a scale that meets worldwide demands, and that also those who would need them most can afford them. MICHAEL KARBIENER 0240343 30 BACCALAUREATE THESIS April & May 2005 ABBREVATIONS A Adenine Asn Asparagine Asp Aspartate C Cytosine cf. confer DNA Deoxyribonucleic Acid e.g. for instance ER Endoplasmatic Reticulum Fuc Fucose G Guanine Gal Galactose Glc Glucose GlcNAc N-acteylglucosamine GTP Guanosinetriphosphate H-bonds Hydrogen bonds Lys Lysine Man Mannose NeuAc Neuraminic Acid = Sialic Acid PMPs Plant-made Pharmaceuticals Pro Proline PTMs Post-translational Modifications RNA Ribonucleic Acid Ser Serine T Thymidine Thr Threonine Tyr Tyrosine U Uracil Xyl Xylose MICHAEL KARBIENER 0240343 31 BACCALAUREATE THESIS April & May 2005 REFERENCES [1] Gomord V, Faye L. Posttranslational modification of therapeutic proteins in plants. Current Opinion in Plant Biology 2004; 7:171-181 [2] Alberts B et al.: Molecular Biology Of The Cell, 4th ed., Garland Science 2002, G:27 [3] Faye L, Boulaflous A, Benchabane M, et al. Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 2005; 23:1770-1778 [4] Spiro RG. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 2002; 12: 43R-56R [5] Bardor M, Faveeuw C, Fitichette A-C, Gilbert D, Galas L, Trottein F, et al. Immunoreactivity in mammals of two typical plant glyco-epitopes, core a(1,3)fucose and core xylose. Glycobiology 2003;13:427-34 [6] Gomord V, Sourrouille C, Fitchette AC, Bardor M, Pagny S, Lerouge P, et al. Production and glycosylation of plant-made pharmaceuticals: the antibodies as a challenge. Plant Biotechnology Journal 2004;2:83-100 [7] Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, et al. Function and glycosylation of plant-derived antiviral monoclonal antibody. Proceedings of the National Academy of Sciences of the United States of America 2003; 100:80138018 [8] Bardor M, Loutelier-Bourhis C, Paccalet T, Cosette P, Fitchette AC, et al. Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a Nglycosylation that is suitable for glyco-engineering into human-compatible structures. Plant Biotechnology 2003; 1:451-462 MICHAEL KARBIENER 0240343 32 BACCALAUREATE THESIS [9] April & May 2005 Arntzen C, Plotkin S, Dodet B. Plant-derived vaccines and antibodies: potential and limitations. Vaccine 2005; 23:1753-1756 [10] Daniell H, Stratfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science 2001; 6:219-226 [11] Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends in Biotechnology 2003; 21:570-578 [12] Ma JKC, Drake PMW, Christou P. The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics 2003; 4:794-805 Fig. 1 http://nobelprize.org/medicine/laureates/1999/illpres/cel.gif Fig.2 http://www.agen.ufl.edu/~chyn/age2062/lect/lect_07/9_15.GIF Fig. 3 Gomord V, Faye L. Posttranslational modification of therapeutic proteins in plants. Current Opinion in Plant Biology 2004; 7:171-181 Fig. 4, Table 1: Ma JKC, Drake PMW, Christou P. The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics 2003; 4:794-805 Fig. 5 Daniell H, Stratfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science 2001; 6:219-226 MICHAEL KARBIENER 0240343 33