Recombinant Proteins - Coursework Example

Recombinant Proteins Introduction Recombinant DNA or rDNA, is the type of DNA that is formed when two or more gene sequences are combined together, artificially, through a process known as ‘gene splicing’ (Berg, Tymoczko & Stryer, 2007). It is different from genetic recombination, as it does not occur naturally within a living cell, but the whole process in engineered by scientists in a laboratory under controlled settings. The chief objective of this process is to modify or alter certain genetic codes, or to correct certain specific traits. It is accomplished by introducing the relevant piece of a DNA strip into the genetic material of another unrelated living organism. When this artificial DNA (rDNA) is inserted into the cell of another organism, the later produces a protein, based on this recombinant DNA type, and this resulting protein is known as the ‘recombinant protein’. In this article I will discuss the process which creates rDNA, and its end product the recombinant protein. My article will take a close look at this recent technological innovation which has conceptualised ‘recombinant proteins’, and explore its importance in the field of biotechnology and in other scientific studies. This article will also take a look into the ethical debate associated with this entire process of producing new genetic codes to create new traits, keeping in view the various advantages obtained from the process of ‘gene modification’, achieved through genetic engineering. Discussion The concept of producing rDNA was first posed by students of the Stanford University, Peter Lobban and Dale Kaiser in 1972. This technique was then further explored and realised by Cohen, Chang, Boyer, & Helling in their paper “Construction of Biologically Functional Bacterial Plasmids in Vitro” published in 1973 (Cohen, Chang, Boyer, & Helling, 1973). Recombinant technology became a reality when in 1970 Daniel Nathans, Hamilton O. Smith, and Werner Arber first recognised and isolated HindII, a restriction endonuclease (they were awarded the Nobel Prize for medicine in 1978). With the successive discoveries of more restriction endonucleases it became possible for the scientists to produce insulin in large scale quantities for the diabetic patients, by using this recombinant DNA technology on the bacteria E.coli. What is recombinant DNA: DNA or deoxyribonucleic acid is the basic hereditary material that stores all genetic information pertinent to the future functioning and development of all living organisms (with few exceptions in the form of viruses). The chief function of this DNA is to store the information that acts like a blueprint for all future developments of a living organism, starting with the coded information necessary for the construction of the basic cellular components, like RNA and proteins. The DNA sequences that store this type of particular information are known as ‘genes’, while the remaining DNA sequences help in regulating this stored genetic information. DNA is located mainly within the nucleus of a cell and is known as the nuclear DNA; however there are small amounts of DNA located within the mitochondrial cell, which is known as the mitochondrial DNA or mDNA. A DNA molecule consists of a double polymer strand, which is composed of simple units known as the nucleotides, having sugar-phosphate backbones tha are held together by ester bonds. Since these two polymer strands run in opposite directions to each other, they are also referred to as the ‘anti-parallel strands’. In the sugar-phosphate backbone, each sugar (deoxyribose) is attached to one particular base molecule. There are 4 different kinds of nitrogen bases as seen within a DNA molecule. These are adenine (A), guanine (G), cytosine (C), and thymine (T) (fig: 1). Coded information is stored in the DNA in the form of these 4 bases. “Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences”( Genetic Home Reference, US National library of medicine, 2010). Fig 1: The double polymer helix of DNA, showing the sugar phosphate backbone and the attached base molecules that from a sort of bridge between the anti parallel strands, creating ladder like structure (Genetic Home Reference, US national library of medicine, 2010). Each base molecule in the DNA strands pair up with only another particular base molecule, to form units that are known as base pairs. Thus, base pairs are formed with adenine always combining with thymine, and cytosine with guanine. Each base in turn is attached to a sugar and a phosphate molecule, and this entire structure comprising of the base pairs and the sugar phosphate backbone is known as the nucleotide. The two nucleotide strands are arranged in the form of a spiral double helix, which looks like a ladder, with the base pairs forming the steps of the ladder, and the sugar phosphate backbone being the two sides (fig 1). Each DNA strand that we see in the double helix can produce exact copies of itself (DNA replication), and this replication is necessary when cells divide, and each resulting daughter cell must necessarily be an exact copy of the mother cell, with the same DNA makeup. When this process of DNA replication is controlled and manipulated externally, it is known as the recombinant DNA or rDNA technology. It involves slicing or excising a relevant DNA sequence from an organism and inserting it into a target plasmid of a bacterial cell. This slicing or excising of DNA sequences is processed with the help of restriction endonucleases, and once inserted into a target plasmid, scientists are able to study the functions of the artificially combined genes, thus being able to comprehend the malfunction/dysfunction of specific genes, right at a molecular level. Recombinant DNA technology: The DNA strands that store information pertinent to all future developments of an organism, however does not actually create an organism. Its chief function is to create protein molecules, and this end is achieved when DNA is transcribed into mRNA, and m RNA translates into the protein molecules. It is these protein molecules that ultimately form an organism. The process of protein formation can be altered, if the DNA sequencing can be modified, artificially or naturally, and the resulting protein or the recombinant protein may either be an inactive one, or of a different variety. Thus the term ‘recombinant’ originates when a DNA sequence from one organism is combined with another DNA sequence from a different organism. This process also often referred to as ‘chimera’, results in the formation of a new DNA sequence, which otherwise would not have been formed under natural circumstances. Recombinant DNA is produced by three methods. These are transformation, bacterio-phage transformation, and transformation of a non-bacterial nature. Transformation: Here a DNA sequence which is to be sliced or excised is first identified, then this marked DNA sequence is cut using a restriction endonuclease, and the sliced DNA is then inserted into the vector with the help of DNA ligase. The inserted piece is given a selectable marker (often antibiotic markers are used, to ensure that only the host cells that have combined with the vector survives), so that one can easily identify the recombinant DNA piece at a later stage. The vector cells are then inserted into the host cell body by transformation, where the host cells are specially prepared so that can receive the new DNA sequence. The selectable markers that were earlier given are now used to trace and differentiate between the transformed cells and untransformed cells within the body of the host. These markers are of various kinds, ranging from antibiotic markers, to those that change colours, or any other variety that can be easily traced within the host’s body. In majority of the DNA transformation, the host cell is a bacterial cell like, that of E. coli. Phage transformation: This is also known as ‘transduction’ and is more or less the same as that of transformation. Here the only difference is that instead of the bacterial cell, a bacteriophage (MI3 or lambda phage) is used. In non bacterial transformation the host cell used is a non-bacterial cell (it may be yeast, or a plant cell, or even an animal cell), and the process may involve ‘electroporation’ where electric shock is given to make transient pores on the cellular membranes of the host cell to allow the entry of the DNA sequence; or, ‘particle bombardment’ where DNA is coated with tungsten or gold particles and then shot with high velocity micro projectiles into the host cell body. rDNA starts functioning only when host cell starts expressing proteins from its recombinant gene sequences. For this recombinant protein production to take place in significant quantities, there must be the presence of certain expression factors, and this protein expression takes place in the presence of particular signals (like the ribosome binding site, the promoter and the terminator) that provide instructions for the processes of transcription and translation of the inserted gene sequence. These signals are specific for each species, and it is highly unlikely that a signal which is specific to the E.coli will be able to translate the signals from a plant, animal or human promoters and terminators. Recombinant protein synthesis: It has been seen that many eukaryotic proteins which are essential for the production of many life saving drugs, and other industrial usages, are found in nature only in very small amounts. It is for this reason various research works on the in-vitro manipulation of DNA sequencing in prokaryotic organisms have been going on for the past few decades, with aims to facilitate the production of these desired proteins, under controlled laboratory environment within the body of a foreign host. The most common form of such protein production for various scientific, industrial and medical purposes is the expression of heterologous genes in bacterial cells. Escherichia coli, gram negative bacteria found in the intestines of human beings, is the most popular variety used for large scale production of recombinant genes, and thus this bacterium plays an important role in the field of industrial microbiology and biotechnology (Lee, 1996). Owing to its versatile and well developed characteristics that allow the scientists to insert genes along with plasmids into the body of the microbe; strong promoters and terminators; and its ability to grow in high cell density; makes it possible for a large scale production of ‘heterelogous proteins’ (Mus-Veteau, 2002), that is required in the process of industrial fermentation. Proteins are organic molecules present in all living organisms, and are essential for the development of all characteristic features of a living body, starting right from the colour of the eye, to the correct functioning of the body’s immunity system. All these are determined by the activities of the protein molecules within the body of that organism. Protein molecules are typically large in nature, and thousands of amino acids form the building blocks for each such molecule. Proteins are formed within a body through a process known as ‘translation’ where the mRNA carrying the genetic code (after the nuclear DNA transcription) enters the cytoplasm, where in the presence of ribosome the process of protein translation takes place. The large scale production of recombinant protein is dependent on many aspects, like the expression factor levels, biological action of the desired protein, characteristics of cellular growth, and both the extracellular and intracellular expression factors, besides the economic factor consideration. Here the bacteria E. coli, plays an important role since it is possible to grow this prokaryote in high scale densities, and coupled with the recombinant DNA technological innovation, it is now possible to produce large quantities of recombinant proteins, at a much lower cost, to be used for clinical and industrial purposes. In the process of recombinant protein purification and expression, 6 or more Histidine molecules act as the metal binding site (these metal binding sites both in recombinant proteins and natural proteins, act as locations for the protein purification process). This hexa-Histidine sequence contains a cleavage site for specific protease molecule, and with the help of certain vectors can be inserted into the N-terminal of a target protein. Then the hexa-histidine recombinant proteins can be purified by the ‘Metal Chelate Affinity Chromatography’ or by using gel beads to produce purified protein, ready to be used for clinical or industrial purposes. The process of producing recombinant proteins using E. coli was a major breakthrough in the field of medical sciences, as it led to the production of the ‘human insulin’, the first clinical product (medicine for the diabetic patients) made through recombinant DNA technology, and approved by the FDA. Human insulin was the first protein to be produced by the scientists, since it had a comparatively simple structure, and making it easy to replicate under in-vitro conditions. In this process first a specific genetic sequence (specifically, an oligonucleotide is used for this purpose; an oligonucleotide is a short polymer of nucleic acids having 20 or less bases) with the necessary codes for insulin production is introduced into the colony of E. coli, and with the life cycle of E. coli being only for about half an hour, within a span of one day there are millions of E. coli bacteria with the coded DNA sequence for human insulin production (Johnson, 1983, 632). Fig 2: Production of recombinant proteins.  (a) The expression vector contains the lacpromoter and its neighbouring lacZ gene encoding b-galactosidase.  Lactose or its analog IPTG will stimulate the expression of b-galactosidase.  (b) If lac Z is replaced by the gene encoding the protein of interest, lactose or IPTG will stimulate the expression of desired proteins (Source: Molecular Biology Web Book, Production of recombinant proteins) Ethics of using recombinant proteins: The advancement in the field of biotechnology, involving the study of DNA and its replication under in vitro conditions, have brought about revolutionary changes in many areas of study, like molecular biology, bio-engineering, agriculture (growing of genetically hybrid crops) and medical sciences. With the help of recombinant DNA technology, it is now possible “to clone the isolated gene and grow it on a large scale, analyse it by sequencing its nucleotides and express it either in bacteria or in mammalian cells in culture to produce large quantities of the protein. The function of a gene can be elucidated by studying the protein it makes. Mutations in certain genes result in defective protein synthesis, thus resulting in a diseased condition....Diagnosis of some of the diseases including genetic disorders, cancer and infections has become swift and...Individuals at an increased risk for a particular disease can be diagnosed at a very early stage in life” (Mulherkar, 1997). However this production of recombinant DNA, or ‘DNA cloning’ as it more frequently referred to, like any other new technological innovation, is facing some ethical concerns from certain scholarly and religious circles, on the issue of producing genetically modified strains that are supposedly ‘unnatural’. The technology of recombinant DNA production involves creation of genetically modified organism or GMO, where the coded DNA sequence of a species is inserted artificially into another species, thus producing a GMO with a completely new DNA strain, which would otherwise have not been possible under natural conditions. This is where the ethical question arises as to whether one has the right to create such selective new strains, giving them preference over naturally occurring varieties. According to the European Commission GMO is defined as “A genetically modified organism is an organism, with the exception of human beings, whose genetic material has been changed in a way that is not possible naturally through reproduction and/or natural recombination”( GMO-Free Additives and Processing Aids for Organic Food and Feed Production, 2008, 8). These GMOs can be used to produce the newly coded protein known as the recombinant proteins, and according the Human Genome Project, GMOs are mainly created in order to produce some sort of beneficial effect on the organism that is being genetically altered. GMOs have shown to produce hugely beneficial effects, in the fields of medicine and agriculture. In the latter field, recombinant proteins have been used in many crops to produce genetically modified strains that produce better yields while being resistant to drought, pests and various diseases. This technology has also been successfully used by the Genome Project to create many life saving drugs (like the insulin), and other important vaccines. However, there are also many risks involved in this process, since the introduction of new genetic strains may yield certain unexpected results. It has been speculated that genetically modified crops have the potential of introducing new allergens in the human food cycle, which may cause allergic reactions in certain people. In genetically modified crops that are growing in the open fields, there are chances that owing to cross pollination by natural pollinating agents there may be created further new genetic strains (and also new recombinant proteins), without the knowledge of the farmers, and the end products of these ‘uncontrolled’ strains may not be entirely desirable. In case of GMMO or genetically modified microorganisms there has not been much breakthrough, “however, a lot of work has been done on GM starter cultures at the laboratory scale...One example is a Lactobacillus that cannot metabolise lactose; as a result, yoghurt does not turn acidic and bitter during storage. A starter culture has also been developed that produces a compound breaking down the cell walls of Listeria. The advantages [are] resistance to phage attack and production of flavour compounds for better taste are other examples of successful modification of starter cultures” (Kärenlampi, 2000, 8). In 1990 England became the first country to permit use of live GMMO in food, where “baker’s yeast engineered to make the bread dough rise faster, by breaking down maltose more efficiently. This modification was done by using the yeast’s own genes and placing them behind a stronger, constitutive promoter” (ibid). Similar methods have been used for beer productions, which have resulted in a variety of beer, which has the full strength of the normal beer with however low calories. The chief concerns in relations to using live GMMO in food, are in relation to the creation of potentially harmful end products that may cause allergy or poisoning, or may have factors that could be ‘antinutritional’, or with adjuvant effects, that may in turn cause inflammatory and auto immune diseases in human beings. There is also the ethical question of giving genetic transfer ‘selective advantage’ over the natural intestinal bacterial cells. However industrial GM producer organisms are widely used in the food processing industry, like Chymosin and Lipase, and no questions regarding their usage have been raised. Conclusion Recombinant DNA technology that produces recombinant protein is indeed a technological marvel in the world of biological sciences. It has helped in the production of many life saving drugs and vaccines; while also creating many crops that are producing better yields, thus helping in assuaging the worldwide food crises and hunger. The ethical question as to whether one has the right to create something that does not occur naturally, can be safely overlooked in this case of recombinant proteins, whose advantages far outweigh the disadvantages (till date only speculations), and in the modern context there is indeed no doubt that the process of recombinant DNA technology has gone a long way in helping mankind to great extent. Bibliography Berg, J., Tymoczko, J., & Stryer, L., 2007.  Biochemistry. San Francisco: W. H. Freeman. Cohen, S., Chang, A., Boyer, H., & Helling, R., November 1973. Construction of Biologically Functional Bacterial Plasmids in Vitro. Proc. Nat. Acad. Sci. USA, Vol. 70, No. 11, pp. 3240-3244. GMO-Free Additives and Processing Aids for Organic Food and Feed Production, 2008. LIS consult, Animal Sciences Group of Wageningen University & Research, 8. Accessed at, http://www.gmfreeireland.org/feed/documents/GMOfree_additives_and_processing_aids_for_organic_food_and_feed_production.pdf Johnson, I., 1983. Human insulin from recombinant DNA technology. Science: Vol. 219. no. 4585, pp. 632 – 637. Kärenlampi, S., 2000. Introduction to genetically Modified foods: Plants and microorganisms. In, Danish Veterinary and Food Administration, “Genetically Modified Foods”. Nordic Committee on Food Analysis, Bornholm (Denmark), 8. Lee, S., 1996. High cell-density culture of Escherichia coli. Trends Biotechnol. 14 (3): 98–105. Mulherkar, R., 1997. Biotechnology and medicine: ethical concerns. Indian Journal of Medical Ethics. Accessed at, http://www.issuesinmedicalethics.org/054mi110.html Mus-Veteau, I., 2002. Heterologous expression and purification systems for structural proteomics of mammalian membrane proteins. Conference review, Comparative and Functional Genomics, Comp Funct Genom; 3: 511–517. Accessed at, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2448422/pdf/CFG-03-511.pdf Fig 1: Genetic Home Reference, 17th October 2010. What is DNA? US National Library of Medicine. Accessed at, http://ghr.nlm.nih.gov/handbook/basics/dna Fig 2: Molecular Biology Web Book, nd. Production of recombinant proteins. Accessed at, http://www.web-books.com/MoBio/Free/Ch9H.htm Read More