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Electrophoresis is defined as the migration of a charged particle in an electric field. Electrophoretic techniques have been used to analyze, separate, and characterize proteins, peptides, and nucleic acids. A number of different one-dimensional electrophoresis techniques have been developed using various kinds of gel, including starch, agarose, or cellulose acetate. A major breakthrough in electrophoresis methodology occurred with the development of polyacrylamide gels (1). Polyacrylamide gels are nonionic polymers, which are chemically inert and stable over a wide pH range, temperature, and ionic strength. Moreover, they allow gels to be prepared with a wide range of pore sizes, facilitating the separation of proteins of different mol wt. A polyacrylamide gel is formed by the polymerization of monomers of acrylamide with monomers of a suitable bifunctional crosslinking agent, usually N,N′-methylene-bis-acrylamide (Bis). The concentration of acrylamide and bis-acrylamide will determine the physical properties of the gel, especially pore size. Polyacrylamide gels are transparent, allowing excellent visualization of the separated proteins after electrophoresis. Polyacrylamide gel electrophoresis (PAGE) can thus be consider as a major protein purification technique, allowing the separation of proteins according to their mol wt, charge, and geometric configuration. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, secreted proteins can be subjected to numerous post-translational modifications. One of these, glycosylation, consists of the addition of sugar residues to the protein backbone, while the protein enters or travels within the secretory pathway. Glycosylation of secreted proteins can be of two types∶ N- or O-glycosylation, depending on the linkage involved between the oligosaccharide moiety and the protein backbone. [ABSTRACT FROM AUTHOR]

An IgG-type antibody is a Y-shaped protein whose arms form two identical antigen-binding sites that are highly variable between different molecules. The stem of the Y is part of the constant region of an antibody and has very limited diversity, which can be used to detect and quantitate antibodies. The interaction of an antibody with its antigen involves multiple noncovalent bonds, such as hydrogen bonds, electrostatic, Van der Waals, and hydrophobic attractive forces (1). The high specificity of this interaction enables an antibody to recognize its antigen even in the presence of a huge amount of contaminating antigens. This makes antibodies indispensable tools in a wide range of disciplines, including molecular biology. [ABSTRACT FROM AUTHOR]

In almost all living organisms, proteolytic enzymes are involved in a variety of cellular functions not only associated with the control of specific endogenous metabolic reactions, but also with the degradation of abnormal or exogenous ("foreign") proteins (1). Despite the fundamental importance of proteases in cells, studies on these enzymes, for those involved in gene-expression technology, are devised to develop a means of avoiding or minimizing degradation of the recombinant proteins to be produced. Some proteins are rapidly degraded either during or shortly after their synthesis, and others are lost during their extraction from cells or tissues. Although general strategies have been proposed to minimize extraction-related hydrolytic processes in microbial, animal, and plant systems (2,3), in vivo proteolysis still represents one of the most significant barriers to recombinant gene expression in any organism (4). Some exo- and endoproteases from Escherichia coli (5) and yeast (6), notably, represent harmful molecules for recombinant ("abnormal") proteins expressed in these systems, and strategies have been developed to counteract potential or actual hydrolytic processes (7,8). Concurrently, the posttranslational ubiquitination of foreign proteins recognized as abnormal in yeast and other eukaryotic cells may lead to their rapid degradation by the multicatalytic complex proteasome via the ubiquitin-mediated proteolysis pathway (9), rendering necessary the study of ubiquitin conjugates when planning to express recombinant proteins in the cytoplasm of yeast or animal cells (10). [ABSTRACT FROM AUTHOR]

We are interested in studying plant systems as vehicles for the production of recombinant proteins of clinical relevance. There are a number of potential advantages to producing recombinant protein products in plants. Plants are more economical compared to fermentation or cell-culture facilities and their production scale can be easily increased. They offer the potential for very high levels of heterologous protein production and because many plant tissues are generally recognized as safe, the probability of health risks because of contamination with potential human pathogens and toxins in recombinant products derived from plants is minimized. Furthermore, plants have glycosylation machinery like other eukaryotic systems (1). [ABSTRACT FROM AUTHOR]

The importance of proteolytic enzymes in plant-pest and plant-pathogen interactions has recently been recognized, and control strategies based on their inhibition with protease inhibitors (PIs) have been developed or proposed to control herbivory insects (1), plant parasitic fungi (2,3), and nematodes (4). The various roles of proteases in these organisms and the biochemical pathways affected by their interactions with PIs may differ, but their importance for normal growth and development is now evident. The repressive effects of PIs on insect growth and fecundity, notably, have been documented for several species (1), and evidence for the implication of microbial proteases as phytopathogenic determinants has been reported in several cases (5-13). Based on this information, transformation of plant genomes with PI cDNA clones appears to be an attractive approach for the control of plant pests and pathogens, and several economically important plants expressing exogenous plant PIs have been engineered during the past few years (Table 1). While allowing control of plant pests and pathogens, PIs expressed in transgenic plants may also serve as a source of active inhibitors for the study and the eventual control of some protease-related pathogenic processes in humans. Proteases are important not only in the intracellular regulation of peptides and proteins, but also in the development of several diseases, including tumor metastasis (21), rheumatoid arthritis (22,23), Alzheimer's disease (24), emphysema (25), pancreatitis (26), and AIDS (27,28). Table 1PI-Expressing Transgenic Plants: Some ExamplesaPlantInhibitorClassRef.RapeseedOryzacystatin ICys 14 bPoplarOryzacystatin ICys 15 bPotatoOryzacystatin ICys 16 RiceOryzacystatin ICys 17 TobaccoCowpea trypsin inhibitorSer 18 bPotato proteinase inhibitor IISer 19 bTomato proteinase inhibitor ISer 19 Tomato proteinase inhibitor IISer 19 bOryzacystatin ICys 20 TomatoOryzacystatin ICys 4 cOryzacystatin I (modified)Cys 4 caActive recombinant inhibitors in planta have been detected in each case.bAdverse effects against growth, development, and/or fecundity of herbivory insects have been demonstrated.cAdverse effects against growth, development, and/or fecundity of the plant parasitic nematode Globodera pallida have been demonstrated. [ABSTRACT FROM AUTHOR]

Photosynthesis, as the basic process leading to biomass accumulation, is intrinsically limited by the performance of the photosynthetic apparatus under different environmental conditions. Potentially, substantial increases in crop yield and improved efficiency of production could be achieved by increasing leaf photosynthetic rates. An essential prerequisite to improving the efficiency of photosynthesis is an understanding of the individual steps involved, their regulation, and interactions with the external environment. Once potential targets have been identified, techniques enabling stable genetic transformation of important crop plants are available to make many of the specific changes we may require. [ABSTRACT FROM AUTHOR]

Genes encoding heterologous proteins are introduced into the plant genome for several purposes. First, the plant-made protein can be used as a tool in fundamental research. Reporter proteins can be used to characterize promoter sequences and other cis-acting sequences; the overproduction of proteins in plants can help to elucidate their function. Second, the synthesis of heterologous proteins in plants can have biotechnological applications. The introduced gene can confer new properties to the plant, or transgenic plants can be used as a source of important heterologous proteins. For all these purposes, the expression profile and accumulation level of the heterologous protein in plants should be stable, reproducible, and suitable. This chapter deals with the following problems∶ Most primary transformants show relatively low expression levels of the transgene (1); and expression levels are not always stably transmitted to the progeny. [ABSTRACT FROM AUTHOR]

The ability to introduce foreign genes into plant species by techniques such as Agrobacterium-mediated transformation or by direct gene transfer (e.g., biolistics) has opened up the possibility of using transgenic plants as host organisms for the production of heterologous proteins (seeref. 1 for an excellent review). Although a number of host organisms (such as Escherichia coli, yeasts, and mammalian cell cultures) are already used for the production of recombinant proteins, plants have several advantages that make them highly attractive for this purpose. For example, the large-scale production and processing of plant material is a routine, cost-effective process carried out by every nation with any form of agricultural industry, thus reducing the need for specialized growth and harvesting equipment. Plants offer additional advantages as potential producers of recombinant proteins. Not only do they modify proteins (in terms of glycosylation, prenylation, and so on) in the same way as other higher eukaryotes (2), but also, since they transmit the transgene encoding the heterologous protein in a Mendelian fashion, the seed of a transgenic plant can be used as the source of further producing lines. This seed also serves as a convenient way of storing biologically viable transgenic material without the need for either cryopreservation or continual passaging/subculturing. The potential offered by transgenic plants has been widely appreciated and anticipated, and a range of innovative strategies have been taken to either alter the endogenous compositions of plant products, or use plants as bio-reactors for the synthesis and accumulation of heterologous proteins. [ABSTRACT FROM AUTHOR]

Transient expression of genes in plant protoplasts is a powerful tool in plant molecular biology that allows quick screening and analysis of engineered proteins prior to stable transformation. Although stably transformed plant material may be preferable in many instances, transformation and regeneration of plants is time-consuming, tedious, and requires plenty of space. Transient expression techniques enable measurement of gene expression, as well as analysis of protein stability and activity, very shortly after DNA uptake; therefore, a large number of samples can be analyzed in a short period of time. Moreover, most of the introduced plasmid DNA remains extra-chromosomal during the transient assay (1) and, consequently, gene activity is not biased by position effects, as observed in stably transformed plants. Because of its speed, convenience, and flexibility, transient gene expression has been widely used for analysis of promoter and regulatory elements involved in transcription and translation (2-6), induction of gene expression by exogenous stimuli (7,8), and for verifying functionality of cloned genes or cDNAs. [ABSTRACT FROM AUTHOR]

The transfer of foreign genes to plant cells is most often performed using the biological vector Agrobacterium tumefaciens (1). This Agrobacterium-mediated introduction of cloned genes into plants has been widely used for promoter characterization using reporter genes, analysis of gene function, and protein targeting. Agrobacterium-mediated gene transfer has also been a method of choice for introducing genes that increase crop resistance to insects or viruses, or genes that encode foreign proteins of industrial or bio-pharmaceutical interest. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (MAbs) (1), because of their binding specificity and stability both in vivo and in vitro, are extremely useful tools in medicine, biology, and organic chemistry. The combination of hybridoma technology and recombinant DNA techniques have given access, not only to full-size molecules, but also to various recombinant antibody fragments (RAbs) and fusion proteins (2), thus broadening the range of possible applications. Recent improvements in heterologous gene expression systems (3) and the development of phage display technologies (2) have made it possible to design and express RAbs against almost any given antigen, and to fine-tune these molecules with respect to improved performance. Furthermore, incorporation of affinity tags has simplified the purification of heterologously expressed recombinant proteins (4,5). [ABSTRACT FROM AUTHOR]

Expression of foreign proteins in plants has become a standard technique in plant molecular biology. Various plant species have been used to produce mammalian proteins, such as human interferon (1) and serum albumin (2), as well as murine antibodies. Not only full-size antibodies (3-6) but also Fab fragments (7) and single-chain fragments (scFvs) (8,9) have been expressed successfully in tobacco orArabidopsis, reaching expression levels as high as 1.3% of the total soluble protein (3). ScFvs have also been expressed in plant-suspension cultures at levels of 0.5% total soluble protein (10). The feasibility of expressing and targeting recombinant antibodies (rAbs) (11,12) has been achieved in different compartments of plants, including the cytoplasm (scFvs), endoplasmic reticulum, chloroplasts, and the intercellular space (full-size, scFvs, and single-domain antibodies) for various applications (3-10). These results indicate the flexibility of the plant system for expression of rAbs, or fragments thereof, in various plant cell compartments. [ABSTRACT FROM AUTHOR]

Plant and animal RNA viruses are increasingly used for gene delivery and expression in a variety of host cells. At a molecular level these agents can be viewed as cytosolic parasites of the ribosome, successfully competing for translation with the host messenger RNA (mRNA) population. In many virus-infected plants, modified plasmodesmata between adjacent cells enable movement of progeny virus throughout the whole organism. Because of this unique virus-host interaction, crop plants provide an inexpensive source of biomass available for rapid genetic manipulations. [ABSTRACT FROM AUTHOR]

Methods of gene cloning and genetic engineering of immunoglobulins and transgenic plant techniques have given rise to new approaches in plant biotechnology (1). The high affinity and specificity of antibodies to different structures can be used to block regulation factors in plant cells by expression of specifically designed antibody constructs (2). Antibodies expressed in plants can also be used to influence and block plant pathogen development and action (3,4). Furthermore, plant cells, especially plant storage organs, can serve as factories for the production of antibodies of biotechnological interest (5). [ABSTRACT FROM AUTHOR]

The discovery that the expression of a viral coat protein in transgenic plants confers protection to infection by homologous and related viruses (1-4) revolutionized the field of plant breeding. The approach of "coat protein-mediated protection" (CPMP) became an extensively studied strategy for many researchers and companies. Coat protein-mediated protection has been demonstrated to be effective against members of more than 10 groups of RNA viruses (3), but the molecular mechanism of the protection still remains unclear (5,6). The phenotype of virus-derived resistance in transformed plants can vary, case-by-case, from a simple delay in normal symptom development, or partial inhibition of virus replication, to complete immunity to challenge virus or viral RNA inoculation. [ABSTRACT FROM AUTHOR]

One of the most important considerations in the expression of heterologous proteins in plants is the choice of promoter. The study of promoter activity is simplified in the majority of cases by the use of a readily detectable reporter gene. Indeed, reporter genes can also be used to aid the isolation of promoters, through the transformation of plants with a promoterless reporter gene construct. Activation of the reporter gene will then only occur if the gene is inserted adjacent to a native promoter (1). [ABSTRACT FROM AUTHOR]

Legumes form an important element in many ecological and agricultural environments. In order to improve the range of phenotypes available for agriculture, plant breeding programs seek to produce improved varieties. Inevitably, these programs are limited by the available gene pool and restricted potential for hybridization between species. Molecular biology techniques offer an opportunity to modify the characteristics of legumes directly. The transgenic plants produced can then be used either in their own right or as a gene pool for inclusion in traditional breeding programs. [ABSTRACT FROM AUTHOR]

Recent developments in genetic engineering and molecular biology have enabled the production of transgenic plants with improved characteristics, or cell and tissue cultures with altered or improved metabolic activity. However, no universally applicable gene transfer method has hitherto been established for plant transformation. The monocotyledonous plants, including grasses and cereals, have proved to be recalcitrant to genetic engineering. They do not belong to the natural host range of the efficient gene vector Agrobacterium, which is widely used for transformation of dicotyledonous plants. Particle bombardment, developed by Sanford et al. (1), is a physical gene transfer method without target material limitations. Using this method, transgenic plants have been produced successfully from all major cereals (2-8). Protoplast-mediated gene transfer methods, e.g., electroporation or polyethelene glycol (PEG) treatment, have long been hampered by the recalcitrance of important cereal crop species to the regeneration of plants from these protoplasts. However, success has recently been reported in this field, even with elite cultivars of barley (9). [ABSTRACT FROM AUTHOR]

Efficient production of transgenic plants is a prerequisite for the production of recombinant proteins in plants. In many dicotyledonous plants, Agrobacterium tumefaciens-mediated transformation has been well established. High efficacy and low cost make this the method of choice in many research laboratories for studying gene expression in plants (1). [ABSTRACT FROM AUTHOR]