2.1 History, nutritional benefits, and availability of peanuts.
The peanut, Arachis hypogaea, is an annual legume rather than a ground nut (Nwokolo 1996). It is an essential crop in the Southern states and is found in a wide diversity of known recipes.
Archaeological records show that peanuts were cultivated in Peruvian desert sanctuary between 300 and 2500 BC (Weiss 2000, Smith 2002). Because of its tropical climate, no archaeological evidences of peanuts were exposed to prove that Gurarani region of Paraguay, eastern Bolivia, and central Bolivia has the greatest diversity of wild varieties of Archis species. It was believed that peanuts were first cultivated in the valleys of Paraguay and Prarane rivers in the Chago region of South America and that peanuts was first to be domesticated by the ancestors of the Arawak.
In 1502, the cultivation of peanut by the Arawak under the name of mani was the first written account of the crop. The name of the crop “mandubi” was first known in Brazil in 1550.
The seeds of peanuts are good source of edible oils that contains 40-50% fat, 20-50% protein, and 10 to 20% carbohydrate. Peanuts are also a good source of dietary fiber and a variety of essential nutrients which includes vitamin B. vitamin E, minerals such as iron, zinc, potassium and magnesium, and antioxidants. Each type of nuts has its own nutritional advantage. Brazil nuts have large amount of thiamin, known as vitamin B1, and an important essential element called selenium.
Peanuts are essential source of non-starch polysaccharides (NSP) with an average 6g/100g of which 1.9g is soluble fiber. NSP plays a significant role in defending the body against bowel disease. Total fiber lowers cholesterol and develops glycaemic control which is important in improving insulin resistance. The high fiber content of peanuts contributes to the satisfying properties of peanuts, compared to other snack foods (Kirkmeyer and Mattes 2000).
In 1900s, it was found out that the regular eating of nuts improves health (Fraser 1999) particularly in lowering the risk of heart disease (Kris- Etheron et. al. 2001). A study conducted by Fraser and his co-researchers (1992) have presented that individuals who eat nut every day had decreased the risk of heart attack up to 60% than those who ate nuts less than once in a month.
Other researches have verified the advantages of eating nuts in reducing heart diseases (Frase 1995; Kushi et. al. 1996; Hu et. al. 1998; Albert et. al. 2002). It was also found that eating nuts have decreased the risk of having a stroke (Yochum et. al. 2000), of developing type-2 diabetes (Jiang et. al. 2002), of developing dementia (Zhang et. al. 2002), of advanced macular degeneration (Seddon, Cote and Rosner 2003) and of gallstones (Tsai et. al. 2004).
Peanut availability is broken down into exports, seed and residual use, peanut crushed for vegetable oil and protein meal, and food use. It has been reported by the National Agricultural Statistics Service that the total supply of peanut in August 2007 to August 2008 is 5,321 million pounds which is the sum of production, imports of shelled and in-shell peanuts, and the total disappearances or the food use is 4,185 million pounds.
2.2 Allergies and Allergens
Allergy is a term which describes body reaction to a substance which is not harmful but causes inconvenience. It is an irritation of the sense of smell, sight, tastes, and touch.
Substances that causes allergy are called allergens. These contain proteins which is often a content of the food we take. Allergens are compound that consists of hydrogen, oxygen, and nitrogen.
Allergy is triggered mostly by environmental factors such as smoke, chemicals, fumes, perfumes, and sometimes physical agents like sunlight, humidity, heat, cold, and haze.
2.2.1 Types of Allergy
Allergies are categorized based on the type of tissue damaged by the allergic reaction. There are four identified types of allergic reaction which includes Type I or Anaphylaxis, Type II or Cytotoxic reactions, Type III or Immune Complex reactions, and Type IV or Delayed-Type Hypersensitivity.
The Type I is IgE-mediated reactions are the most treacherous. The occurrence of IgE antibodies to penicillin and cephalosporin is predictive of probable IgE-mediated, allergic hypersensitivity reactions. IgE produce such reaction. Cytotoxic IgG antibody reaction (type II) and antigen-antibody (IgG or IgM)-mediated (type III) reactions are caused by β-lactam antibiotics, and are hard to predict and these are not allergic reactions. The type II reactions is caused by the intervention of IgG and IgM at the cell surfaces which results to the lysis of blood cells due to the release of complement while the Type III is caused by the intervention of IgG and IgM in fluid spaces that produces toxic antigen-immunoglobulin complexes that circulate in the blood and the complexes adhere to the blood vessel walls and initiates an inflammatory response. The presence of type II or III reactions should direct to prevent the use of drugs (Mendelson 1998). The Contact dermatitis or the Type IV reaction are reactions between antigens and sensitized antigen-specific T lymphocytes. The reaction releases inflammatory and toxic substances and lymphokines that attract other white cells.
2.2.2 Prevalence and statistics on allergy
2.3 Food Allergies
Food allergy is an unusual response to food that is caused by a response in the immune system. The undesirable reactions to food can be sub-divided into non-toxic and toxic.
Food allergens may be antibodies in the blood or cells in the immune system. The antibody responsible for abrupt allergic reactions after eating food is the IgE-antibody. Allergic reactions caused by immune cells or T-cells have delayed reactions.
2.3.1 Causes and symptoms of food allergy
A number of foods are responsible for the majority of food-induced allergic reactions: milk, egg, peanuts, fish, and tree nuts in children and peanuts, tree nuts, fish, and shellfish in adults. Food-induced allergic reactions are responsible for a variety of symptoms involving the skin, gastrointestinal tract, and respiratory tract and may be caused by IgE-mediated and non-IgE-mediated mechanisms. Immuno-pathogenic mechanisms and clinical disorders of food allergy are described (Sampson 1999).
2.3.2 Prevention and Treatment
Patients with food-induced allergic disorders may be first seen with a variety of symptoms affecting the skin, respiratory tract, gastrointestinal tract, and/or cardiovascular system. The skin and respiratory tract are most often affected by IgE-mediated food-induced allergic reactions, whereas isolated gastrointestinal disorders are most often caused by non-IgE-mediated reactions. When evaluating possible food-induced allergic disorders, it is often useful to categorize disorders into IgE- and non-IgE-mediated syndromes. The initial history and physical examination are essentially identical for IgE- and non-IgE-mediated disorders, but the subsequent evaluation differs substantially. Proper diagnoses often require screening tests for evidence of food-specific IgE and proof of reactivity through elimination diets and oral food challenges. Once properly diagnosed, strictly avoidance of the implicated food or foods is the only proven form of treatment. Clinical tolerance to food allergens will develop in many patients over time, and therefore follow-up food challenges are often indicated. However, a number of novel immunomodulatory strategies are in the developmental stage and should provide more definitive treatment for some of these food-induced allergic disorders in the next several years. (Sampson 1999).
Different tests are used to determine the different conditions of the reactions (Table 2.1).
Table 2.1. Test for the presence of allergic sensitization and identification of offending allergens (Lopata 2006).
In 2003, studies have developed an original quantitative enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgE to peanut protein extract and to Ara h 1 and Ara h 2 in a large population of allergic children to peanut, classified in five groups and subgroups depending on the severity of their clinical symptoms. Specific IgE response may allow a first discrimination between patients according to the severity of the symptoms and between allergens according to their prevalence and potency (Bernard et. al. 2003).
During an immune reaction to a foreign antigen, antibodies are produced as part of the body's complex defense mechanism. Antibodies of the IgE type are typical in type I allergic reactions; however, high amount of antigen-specific IgG and IgA antibodies are also observed. In autoimmune disorders, these antibodies are directed against self-antigens (autoantigens). The presence and level of specific IgG antibodies in serum can reflect the extent of exposure to the antigen. IgG antibodies can be quantified via the ImmunoCAP system or the micro-arrays system The IgG antibody response can be quantifies to all available immunoCAP allergens; however, only a few allergens have been evaluated and respective cut-off values determined (Lopata 2006).
2.3.3 Prevalence and statistics on food allergy
Allergy is a rising health problem in the society at all age groups. Allergies to milk, eggs, peanuts, soy and wheat have affected almost 8% of infants and children and 2% of the adult population (Sampson 1999).
In Malaysia, it is estimated that one out of three Malaysians is currently suffering from allergy and it is being expected that in 2020, half of the population in Malaysia will be allergic if the current trend will continue. According to Malaysian Society for Allergy and Immunology, it is estimated that asthma affects 300 million people world wide and an expectation of another 100 million in 2025.
2.4 Peanut Allergy
Peanut allergy accounts for the greater part of acute food-related allergic reactions. It is likely to be present in childhood and its trace quantities can induce an allergic reaction in highly sensitized people (Al-Muhsen 2003).
Peanut allergy is distinguished by more acute symptoms than other allergies and by high rates of symptoms on minimal least contact. A survey on 622 self-reported allergic subjects showed that 66% or 406 individuals have described symptoms on contact with peanuts and only 19% or 121 individuals had been exposed to peanuts before the reaction which implies high frequency of occult sensitization (Weisnagel 2008).
Studies have shown that prevalence of peanut allergy in children is increasing and approximately 0.5 to 1% of the population is affected (Sicherer et. al. 2003). It is estimated that as many as 150 deaths occur each year due to food allergy, and that a majority of these are due to peanut allergy (Bock et. al. 2001). Also, it was found out by Fleischer and his co-researchers (2003) peanut allergy is usually life long although 20% of the patients lose their sensitivity. Those who lose sensitivity can have recurrence that is associated with continued avoidance of the allergen.
A study conducted by Tariq and his colleagues in determining the prevalence of sensitization to peanuts and tree nuts in all children on the Isle of Wight showed that IgE mediated allergy to peanuts is common in early childhood and allergy persists in many but a minority may develop tolerance.
Peanut allergy is more frequent in children than in parents. The evident prevalence may indicate a general increase of atopy, which is inherited from the mother. Peanut allergy is presenting earlier in life, probably reflecting increases consumption of peanut by pregnant and nursing mothers (Hourihane, Dean, and Warner 1996).
Abrupt hypersensitivity to peanuts is a common cause of anaphylactic reactions and deaths in children and adults. At present, preventive treatment consists of avoidance and it is difficult because of its prevalence. Studies showed that injections of peanut extract increase the tolerance of patients with peanut allergy to oral ingestion of peanuts. Injections result in repeated systemic reactions in most patients, even during maintenance injections (Nelson et. al. 1997).
Peanuts are also one of the most common foods responsible for food-induced anaphylaxis. Patients rarely lose sensitivity to peanuts. Although the ideal treatment is avoidance, this is often not possible because of hidden exposures; therefore, a more effective treatment is needed. Subjects with confirmed peanut allergy were treated in a double-blind, placebo-controlled study with peanut immunotherapy or placebo (Oppenheimer 1992).
Anaphylaxis refers to the rapid development of generalized reactions such as pruritus, urticaria, angioedema, hypotension, wheezing, bronchospasm, nausea, vomiting, abdominal pain, diarrhea, uterine contractions and direct cardiac affects including arrhythmias (James 1996). The frequent initial set of symptoms of anaphylaxis includes a sense of threat and generalized warmth which is characterized by pruritus or tingling of the skin, palms of hands, soles of the feet, lips, and also on the genital area. Significant signs that needs an immediate action includes lump in the throat, throat tightness, hoarseness, difficulty in swallowing, inspiratory stridor, chest tightness, wheezing and shortness of breath. The symptoms frequently occur within minutes of exposure but it may not appear after an hour of exposure.
Some of the common features related to the different types of anaphylactic reactions are (1) food-related anaphylaxis which is frequent in younger children with a previous history of eczema an the symptoms usually affect the gastrointestinal system, (2) exercise-induced anaphylaxis that occurs in children with a previous history of ucticaria-angioedema, and (3) venom-induces anaphylaxis that occurs to individuals with negative skin prick tests to common inhalant and food allergens (Elio et. al. 1998).
Epinephrine is a drug used in treating anaphylaxis caused by peanut allergy. Self-injectable epinephrine can be prescribed to patients with anaphylaxis. One's the patient's condition is normalized with epinephrine, diphenyhydramine and ranitidine may be started
2.5 Plant food allergens and its super families
Anaphylaxis (ANA) episodes are the most common causes of food allergic reactions. It was found out that most food allergens belong to protein families. Allergenicity of a particular food depends on membership on certain protein families, stability during food processing and G-I digestion after ingestion, and abundance in the food supply and related to exposures of human (AAAAI 2005).
It was identified by the AAAAI (2005) that three plant protein families contain most plant food allergens. These are the Prolamins, Cupins and the Bet V1 Family Proteins.
The Prolamin family of proteins contains amino acids praline and glutamine which are resistant to proteases and destruction during cooking. The prolamin super family comprises three main groups of food allergens consisting of 2S albumins which contains two subunits that are storage protein in dicot plants, lipid transfer proteins which are monomers that have four disulfide bonds and accumulate in the outer epidermal layer of plant organs, and alpha-amylase and protease inhibitors that provides plants with resistance to insects' and fungi's alpha amylases.
The Cupin family of proteins is a diverse proteins that can be found in bacteria, plants, and animals. A set of 2-domain cupins is a common seed storage protein which is a major component of human diet. The allergic members of the cupin family are the vicillins, which is the major allergens present in peanut, soybean, lentil, pea, English walnut, cashew and sesame seeds, and the legumins which is also present in peanuts, soybean, hazelnut, walnut, almond, Brazil nut cashew and coconut.
The Pathogenesis Related - 10 proteins occur in a wide variety of plants and serve as steroid hormone transporters. These proteins are relatively unstable to heating and digestion and, as a result, tend to cause allergic reactions that are limited to the oral cavity. Primary sensitization to allergens in this family often occurs from the airborne birch allergen Bet V1. The surface epitopes of Bet V1 and related proteins show extensive homology and are responsible for the fruit-vegetable-pollen cross-reactive syndromes.
2.6 Peanut Allergens
Peanut kernels have many allergens able to obtain IgE-mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients identified variable patterns of IgE-binding proteins. The recognition of the IgE-binding proteins of peanut extract would aid improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the recognition of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products.
Some allergens were recognized to be the most frequent cause of peanut-specific IgE in individuals with peanut allergy. It was found out that Ara h 2 is the most common causative allergen for symptom-triggering and generated a response at a moderately low concentration. Ara h 1 and Ara h 2 were known to have be less frequent and only responded to reaction at a higher concentration than Ara h 2. It was concluded that Ara h 2 is the most essential allergen and the others may be significant but with less frequency at a higher concentration (Wensing et. al. 2004)
Some of the peanut allergens are Ara h 1 (a 63.5 kD protein, a vicilin), Ara h 2 (a 17.5 kD protein, a conglutin), Ara h 3 (an 11S globulin seed storage protein family member, a glycinin), Ara h 4 ( a seed storage protein), Ara h 5 (a 15 kDa protein, a profiling), Ara h 6 ( 2S albumin) and Ara h 7 (2s albumin).
Ara h1, an abundant peanut protein, is recognizedby serum IgE from >90% of peanut-sensitive individuals. It hasbeen shown to belong to the vicilin family of seed storage proteinsand to contain 23 linear IgE binding epitopes (Shin et. al. 1998).
Studies have also claimed that Ara h 1, Ara h 2, and Ara h 5 are major allergens, while Ara h 6 and Ara h 7 are minor allergens (Joneja 2007).
While Ara h 1 and Ara h 2 are considered as major peanut allergens, they were not capable to generate PLN cell proliferation or cytokine production, proposing that they contain little intrinsic immune-stimulating capacity. However, PE induced a strong PLNA reaction and was able to produce T cell responses against the allergens present. It was also found that Ara h 1 and Ara h 2 are poor IgG and IgE inducers (van Wijk et. al 2004).
Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. The characterization of the two peanut allergens Ara h 1 and Ara h 2 have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions (Rabjohn et. al. 1999).
Recombinant allergens, which are genetically engineered isoforms resembling allergen molecules from known allergen extracts, have immunoglobulin E (IgE) antibody binding comparable to that of natural allergens and generally show excellent reactivity in vitro and in vivo diagnostic tests.
2.6.1 Peanut Allergen Ara h2
Among theallergenic proteins, Ara h 2 is one of the most commonly recognizedallergens. Ara h 2 is a 17-kDa protein that has eight cysteineresidues that could form up to four disulfide bonds. Circulardichroism studies showed substantial changes in the secondaryand tertiary structures of the reduced Ara h 2 as compared withthe native protein. Upon treatment with trypsin, chymotrypsin,or pepsin, a number of relatively large fragments are producedthat are resistant to further enzymatic digestion.
The Ara h 2 protein does notform any higher order oligomeric structures with itself, butdoes contain eight cysteine residues that have the potentialto form up to four disulfide bonds. Studies have also shown that the overall structure of Ara h 2 is changed when disulfide bonds are reduces and the structure was not completely randomized when the disulfide bonds are reduced but is predominated by a -pleated sheet and -turn configuration.The study have that Ara h 2 is a very ordered protein even without disulfide bonds and it is susceptible to rapid digestion with pepsin, chymotrypsin, or trypsin, indicating a reduction in its overall allergenicity. (Moon et. al. 2002).
Studies have shown that Ara 2 did not always have the same allergenic potency as compared with its natural counterpart. The partial digestion yielded stable immunologically active core structures. Even though the IgE antibody-binding assay showed reduction of antibody-binding capacity, the functional assay (mediator release from a functional equivalent of mast cells or basophils, the humanized RBL cells) demonstrated that reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency (Lehmann et. al. 2006).
Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics (Glenting et. al. 2007).
The ability of Ara h 2 to cross-link EgE effectively from a specific serum is important in peanut allergen and is related to the proportion of an anti0Ara h 2 in that serum but it does not account for a majority of the effector activity of the CPE for any of the sera studied (McDermott 2007).
2.7 Recombinant Protein Production and its relevance in society and in the industry
Recombinant proteins are made from cloned DNA sequences that encode and enzyme or protein with known function. Special tools for manipulating DNA are used for the production of the proteins from specific DNA sequences cloned into them. Because purification of a protein can be complicated and a long process, it is likely to add a DNA sequence to the cloned protein's code that will add a small peptide or a small protein for aiding the purification of the recombinant protein after it is expressed.
Recombinant allergen production can ease some of the problems related to allergen products based on natural sources. Recombinant allergens represent an important tool in component resolved allergy analysis, development of engineered hypoallergens, and production of allergens with defined composition and purity.
Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics (Glenting et. al. 2007).
2.8 Codon optimization
Codon optimization is an experimental method wherein codons contained by a cloned gene or ones that is not usually used by the host cell translation system are altered by in vitro mutagenesis to the desired codons, without altering the amino acids of the synthesized protein.
Amino acids in protein sequence are symbolized by a 3-letter word (codon) in the genetic code. Given that there are 4 letter (A, C, G, T), there are 64 potential words to represent 20 amino acids, and stop codons. Each codon denotes a single amino acid and majority of the amino acids are characterized by multiple codons.
Many proteins are hard to express outside their unique frameworks. The proteins may have expression-limiting regulatory factors that may be organisms that utilize non-canonical nucleotide codes or from a predominant gene with codons seldom used in the desired host. Development in the speed and efficiency of gene synthesis had provided possible complete gene redesign for maximum protein expression.
Codon optimization has been found as the definite most significant in prokaryotic gene expression. The level to which a codon occur in the genetic code differs significantly between organisms, between proteins expresses at high and low levels and even between different portions of the same operon. This is because favored codons associate with the abundance of similar tRNAs present in the cell. This relationship functions to optimize the translational system and to balance codon concentration with iso-acceptor tRNA concentration. For example, in E. coli, the tRNA molecule that translate the rarely used AGG and AGA codons for arginine exist only at very low levels. It is possible that codon usage and tRNA isoacceptor concentrations have coevolved, and that the selection pressure is more pronounced for highly expressed genes than genes expressed at low levels.
A study had demonstrated that codon optimization is a helpful method for improving foreign antigen expression in rBCG and for attaining significant levels for foreign antigen-specific immune responses. This methos is key to rBCG-HIV vaccine development, since low-dose immunization and immunization with 0.1 mg of codon optimized rBCG has proven effective for induction of HIV-specific cellular immunity by allowing for a smaller dosage of rBCG, one that is far more practicablefor use in human tuberculosis vaccination than the 1 to 10-mgdose otherwise required, and by reducing the risksassociated with high-dosage cutaneous administration, includingadverse local skin reactions, possible association with Th2-typeimmune responses, or exacerbation of retroviral infections.Given these results, rBCG is clearly poised to play a key rolein the development of an HIV/AIDS vaccine (Masaru et. al. 2005).
A new codon optimization web server is the “optimizer” which is concentrated on maximizing the gene expression level through the optimization of codon usage. Its sole features includes a novel definition of a group highly expresses genes from more than 150 prokaryotic species under translational section, and the possibility of using information on tRNA gene-copy number in the optimization process. The meth0d gives several pre-computed tables to specify a reference set and merges three different methods of codon optimization. It is also used to optimize the expression level of a gene in heterologous gene expression or to design new genes that present new metabolic capabilities in a particular species (Pare et.al. 2007).
Codon usage is the most significant factor in prokaryotic gene expression. .
The frequencies that distinctive codons are used vary essentially between different organisms, between proteins stated at high or low levels with the same organism. The most probable reason for this variation is that chosen codons are associated with the abundance of cognate tRNAs present within the cell. Because of the design of synthetic genes, the use of codon to encode different amino acids was chosen to suit expression host.
2.9 Cloning vectors
Cloning vectors are DNA molecules that originates from a virus, a plasmid, or the cell of a higher organism to which another fragment can be integrated without loss of the vector's ability for self replication. Some of the vectors cloning include plasmids, cosmids, yeast artificial chromosomes (YACs), phage, and bacterial artificial chromosomes (BACs).
2.9.1 Popular Cloning Vectors
TA cloning is one of the most common methods of cloning the amplified PCR product using Taq and other polymerases that lacks 5'-3' proofreading activity and have the ability to add adenosine triphosphate residue to the 3' ends of the double stranded PCR product which overhangs to each end of the PCR product. Thus, the PCR product can be directly cloned into a linearized cloning vector that have single base 3'-T overhangs on each end and the vectors are called T-vectors and the PCR product with a overhand is combined with T-vectors in high proportion to form a recombinant protein which is brought about by DNA ligase.
Topo cloning is a method that is used to increase a small amount of DNA . The procedure is done by cloning the fragment into a vector, converting E. Coli cells, and using blue or white selection to identify transformants. When a transformant has been separated, a culture can be immunized.
The main element important in Topo cloning is the enzyme, DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. The biological role is to cut and reconnect DNA during replication. Vaccinia virus topoisomerase I particularly identify the pentameric sequence 5'-(C/TCCTT-3' and forms a covalent bond with the phosphate bond with the phosphate group of the 3' thymidine. It cuts one DNA strand, enabling the DNA to unwind and the enzyme then transfer the ends of the cut strand and releases itself from the DNA.
Plasmids are circular, double stranded DNA molecules that are detached from a cell's chromosomal DNA which is present in bacteria, yeast, and other eukaryotic cells that exist in a parasitic or symbiotic relationship with the host cell. Plasmids range in size from a few thousand base pairs to more than 100 kilobases (kb.Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell (Lodigh et. al. 2000).
The plasmids most commonly used in recombinant DNA technology replicate in E. coli. Usually, these plasmids have been engineered to optimize their use as vectors in DNA cloning. For example, to simplify working with plasmids, their length is reduced; many plasmid vectors are only ≈3kb in length, which is much shorter than in naturally occurring E. coli plasmids. (The circumference of plasmids usually is referred to as their “length,” even though plasmids are almost always circular DNA molecules.) Most plasmid vectors contain little more than the essential nucleotide sequences required for their use in DNA cloning: a replication origin, a drug-resistance gene, and a region in which exogenous DNA fragments can be inserted (Lodigh et. al. 2000).
2.9.2 pGem-T Easy Vector
The pGem-T Easy Vector systems are suitable systems for the cloning of PCR products, construction of unidirectional nested deletions with the Erase-a-Base System, Production of ssDNA, blue or white screening for recombinants, and in vitro-transcription from dual-opposed promoters (Promega 2007).
2.10 Different expression systems
Expression systems are aimed to produce many copies of a desired protein within a host cell. In order to achieve this, an expression vector is inserted into a host cell. This vector contains all of the genetic coding necessary to produce the protein, including a promoter appropriate to the host cell, a sequence which terminates transcription, and a sequence which codes for ribosome binding (Purves et al., 2001).
Protein expression system requires a template, a means of transcription such as a cell or cell extract, and the raw materials needed to build proteins. Protein expression system has many types which is dependent on the protein to be expressed and needed (Smith 2007).
Yeast had been genetically characterized and known to complete posttranslational modifications. The eukaryotic cells grow rapidly in defined medium and are easier and less expensive to work than mammalian cells. Yeasts are also adapted to fermentation easily. The system is ideal fro large-scale production of recombinant eukaryotic proteins.
One of the major advantages of yeast expression system is that yeast culture can be grown to very high densities that make them useful for the production pf isotope. Other advantages are high yield, high productivity, chemically defined media, product processing similar to mammalian cells, stable production strains, durability, and lower protein production cost.
The cell-free expression system makes use of effective, coupled transcription and translation reaction to make up to milligram quantities of active recombinant protein. The system removes the time-consuming methods of cell-based protein production such as transformation, cell culture maintenance, and expression optimization steps
The advantages of insect expression system include high expression levels, ease of scale-up, production of proteins with posttranslational modifications and simplified cell growth. Insect cells do not need carbon dioxide for plant growth and can be altered to high-density suspension culture for large-scale expression.
The most popular host organisms for expressing recombinant proteins are the E. coli and mammalian cells.
The bacterial protein expression system is the most common and most economical method of protein expression that is offered at present. But there are two major problems in using the system: (1) the protein to be studied cannot always be expressed using specific vectors and (2) the proteins are sometimes insoluble
Bacterial cell-free protein synthesis is a simple method where DNA is transcribed and translated in vitro to produce protein. The cell-free protein synthesis has advantages over cell-based systems specifically in the expression of toxic proteins, labeling of amino acids for structural studies and expression of mutants of a protein for rapid analysis. Cell-free protein synthesis allows addition of detergents, chaperones and appropriate ligands during the process of protein synthesis, which may aid in proper folding of the proteins
Most of the genes cloned into bacterial expression vectors with T7 promoter, can also be a template for bacterial cell-free expression, obviating the need for sub-cloning. Cell-free protein synthesis entails several ingredients such as tRNA, amino acids, nucleotides, components of energy regenerating system, small molecules and T7 RNA polymerase in optimum proportions. Use of this complex mixture requires extensive optimization to produce proteins in a reproducible manner. Commercial extracts for protein synthesis are highly expensive, not practical for high-throughput studies and are not amenable to modifications, as the composition is not disclosed. (Murthy et al., 2004).
2.11 E.coli expression system
Most cloning experiments are performed with E. coli as the host, and the broadest diversity of cloning vector are available for this organism. E. coli is mostly known when the objective of the cloning experiment is to study the basic features of molecular biology that includes gene structure and function.
The E. coli expression system lets rapid expression and succeeding large-scale, cost-effective manufacturing of recombinant proteins. This system is perfect for antigen expression and functional protein expression for non-glycosylated proteins.
Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system.Under those conditions, the tripartite sensor kinase ArcB undergoesautophosphorylation at the expense of ATP and subsequently transphosphorylatesits cognate response regulator ArcA through a His Asp HisAsp phosphorelay pathway. In this study we used various combinationsof wild-type and mutant ArcB domains to analyze in vitro the pathwayfor signal decay. The results indicate that ArcA-P dephosphorylationdoes not occur by direct hydrolysis but by transfer of the phosphorylgroup to the secondary transmitter and subsequently to the receiverdomain of ArcB (Dimitris et. al. 1998).
The successful expression of any foreign fusion protein in E. coli is dependent on the multiple results which may result in having high yields of a soluble protein (Saluta and Bell 2008).
