Industrial Scale Production of Heterologous Proteins - Essay Example

INDUSTRIAL SCALE PRODUCTION OF HETEROLOGOUS PROTEINS By Location The advances made in genetics in the recent years have made it possible for the production of some of the critical proteins in an industrial scale. For a long time, scientists have focused on understanding the process of protein synthesis in a diverse range of cells. Evidently, the need for proteins of significant importance, such as insulin, human growth factor, somatostatin, albumin, and heparin and virus antigens in emerging medical therapies has prompted the development of procedures of production in high quantities. Evidently, the understanding of gene expression in different cells has been a major benchmark in the production of these proteins. These proteins been have been categorized as heterologous proteins, because of the unique structure that comprises of both multimeric and monomeric forms. Different expression systems have been developed, and the choice of the most relevant expression system depends on the protein of interest. The sequencing of the gene responsible for coding for the protein of interest is critical prior to the production process. Notably, genetic recombinant techniques are handy in the production of heterologous proteins because the gene coding for the protein of interest must be incorporated into the genetic sequence of the host cells. After the successful transformation of the host cells with the gene of interest, placing of the cells in appropriate culture conditions follows. During culture, the cells register growth. As the expression of the host genes takes place, the introduced genes undergo expression as well. Notably, successful transcription and translation are critical for the protein of interest to be produced (Marisch et al 2013, p.14). Moreover, heterologous proteins exhibit a remarkably high molecular weight, and require post-translational modifications and effective folding into active proteins. These processes are of critical significance as they determine the success of any industrial scale venture that aims at producing heterologous proteins. This paper will discuss all the aspects mentioned in depth, highlighting the factor that affect each of the steps involved in the production of heterologous proteins. After the successful production of the target protein, the last process involves rigorous purification of the protein. Main steps in DNA recombinant technology Available at http://journals.iucr.org/d/issues/2005/10/00/ic5044/ic5044fig3.html The initial step in the production of heterologous proteins involves understanding the properties of the protein of interest. This is critical as it determines the appropriate expression system that proves potential for the production. Special consideration is accorded to the molecular weight of the protein of interest, its primary, secondary, and tertiary structure, and the process involved in the protein folding (Idiris et al 2010, p. 408). Understanding the properties of the protein offers an overview of the necessary process, and highlights some of the critical factors that need consideration for effective production of the protein. After understanding the protein of interest, it is critical to consider sequencing the gene coding for the protein. Evidently, without successful sequencing of the gene responsible for coding the protein, it is impossible to produce the desired protein. In the recent decades, geneticists have made remarkable progress in sequencing the genes coding for many of the heterologous proteins considered of critical interest in medicine and other fields. With such success, a new realm of possibilities regarding industrial scale production is open (Idiris et al 2010, p. 410). After successful sequencing of the gene responsible for coding for the protein of interest, recombinant DNA technology, techniques are used in the transformation of the host cells. However, prior to the recombinant technique, it is critical to select the most appropriate expression system, depending on the protein of interest. Choice of Expression Systems Various expression systems have been described about the production of heterologous recombinant proteins. One of the preferred expression systems is the use of bacterial cells. Lactic acid bacteria and Escherichia Coli define some of the outstanding strain used in the production of recombinant proteins (Mierau et al 2005, p.8). With an increased understanding of gene expression in bacteria over the years, as well as the existing expertise of culturing such prokaryotic cells, there is a wide usage of bacterial cells in the production of heterologous recombinant cell. Evidently, researchers have described certain merits associated with the use of bacterial cells as preferred expression systems in some processes of protein production. Without doubt, the ease with which the bacterial genome undergoes successful manipulation is one of the salient merit. Moreover, with emerging new expertise in batch, fed batch, and continuous culture systems, it has proved remarkably easier to culture bacterial cells in high quantities. This increases the possibility of producing high yields of the desired protein. Evidently, there is a remarkable understanding of the culture conditions for bacterial cells. Moreover, geneticists have highlighted the availability of numerous plasmid vectors that serve critical roles in bacterial transformation prompt the choice of bacterial cells as the preferred expression systems. However, despite these salient benefits, bacterial cells fail to meet certain criteria in the choice of a favourable expression system. Research has revealed that bacterial cells are unsuitable for the expression of heterologous proteins of high molecular weight. Moreover, bacterial cells have exhibited the tendency to detect eukaryotic proteins as foreign, and consequently initiate a process of degrading the expressed protein (Tian & Sun 2011, p. 7). Notably, bacterial cells lack the processes involved in post-translational modifications of the produced proteins, and hence are unable to yield active heterologous proteins that are properly modified. In addition, bacterial cells are unable to carry out effective folding of the proteins of interest. Recent research has indicated that bacterial cells exhibit the inability to express eukaryotic genes that comprise of introns. Evidently, introns need removal, as they are not subject to expression. Yeast as expression system In addition to bacterial cells, geneticists have identified yeast cells as viable expression systems that present multiple benefits when compared to bacterial cells. One of the outstanding benefits of using yeast cells is the ease of manipulation of yeast DNA. Recent research has enabled a successful isolation of yeast promoters and naturally occurring plasmids that make the transformation of yeast cells easier (Hahn-Hägerdal 2005, p. 62). Moreover, the food and drug administration (FDA) of the United States has recognized the use of yeast cells as a safe expression system. Despite this benefit, yeast cells also present undesirable attributes that make it impossible to produce active heterologous recombinant proteins. One of these attributes is the act that yeast cells are lower prokaryotes hence lack the appropriate mechanisms that would carry out effective post translational modifications. The result of this is a yield of inactive heterologous recombinant retains. Similar to bacterial cells, yeast cells are also unable to remove introns in genetic sequences of the desired heterologous proteins. This makes the transition and translation faulty. Insect cells as expression systems In the recent years, research has revealed that insect cells are also potential expression systems in the venture to yield high quantities of heterologous proteins. For successful use, if insect cells are used as expression systems the baculovirus vectors are highly preferred. There is evidence of successful use of the baculovirus vectors in large-scale production of recombinant heterologous proteins such as malarial proteins, HIV 1 envelope proteins, polio virus proteins, interferons, bovine rhodopsin, erythropoietin and lesser virus proteins. (Papazyan & Taverna 2013, p.5) The success of the production of these proteins has resulted from the salient potential of the baculovirus to infect and multiply in insect cell cultures. Benefits of using insect cells as expression system have been described in recent research. One of this is the fact that insect cells have the potential to recognize vertebrates, protein codes when in incorporated during DNA recombinant techniques. This means that using insect cells, it is possible to produce active heterologous proteins because successful post translation medication occurs. The heterologous proteins are either available in the media or sometimes secreted into cultured insect cells. Moreover, insect cells have higher eukaryotes compared to yeast cells and therefore present more benefits. However, researchers have not managed to understand the manipulation of insect DNA in depth posing a major challenge in the DNA recombinant techniques (Branco et al 2008, p. 74). Mammalian cells as expression systems Evidently, mammalian cells present the most preferable expression system in the venture to produce high quantities of heterologous recombinant proteins. Since most of the heterologous proteins of interest are actually synthesized in various mammalian organs, using mammalian cells presents multiple benefits compared to all other expression systems discussed above. One of the obvious benefits is the fact that mammalian cells have the required mechanisms involved in post translational modifications (Postis et al 2013, p. 43). This offers an assurance of the production of active heterologous proteins. Notably, mammalian cells recognize similar signals, making it possible for them to carry out effective transcription and translation yielding the heterologous protein of interest. For a long time, the challenge of using mammalian cells was identifying effective vectors for use in the recombinant DNA technology, which is critical in transforming the mammalian cells for use in protein production. An additional problem was the limited understanding of the mammalian genome. This placed a salient need for sequencing many of the genomes of mammals (Tingfeng,Yuansheng, & Kong 2013, 601). However, increased research in the past decade has presented a new realm of the use of mammalian cells as expression systems. This is because of successful research in identifying the following: Efficient promoter elements Numerous Plasmid sequences Multiple expression vectors With success in the above mentioned aspects the use of mammalian cells as expression systems has proved viable. The availability of both versatile and effective mammalian expression vectors has resulted in the production of high quantities of heterologous recombinant proteins that exhibit the highest levels of authenticity. Such proteins have all the required post translational modifications in place and exhibit a high level of activity. However, there is a need for new concepts and techniques that will serve to minimize the high cost of using mammalian cells as expression systems. Notably, there is limited understanding of the appropriate culture conditions of optimized growth of the mammalian cells for muse in protein production (Nozawa et al 2011, p. 35). DNA recombinant techniques The discovery of multiple restriction enzymes and their significant application in cleaving different DNA sequences has been a benchmark in the success of the recombinant DNA technology. Evidently, it is possible or a gene of interest to be incorporated into a selected genome because of the potential DNA cleavage achieved by restriction enzymes. The first step in the production of heterologous recombinant proteins involves sequencing of the gene responsible for the coding of the protein of interest. After successful sequencing, the gene of interest is inserted into a selected vector. Selection of an effective vector depends on multiple factors. However, the most critical factor is the expression system chosen (Lilie et al 2013, p. 1000). Notably, vectors vary between the different expression systems described above. Therefore, a research should have an in-depth understanding of the appropriate vectors for use in the selected expression systems. Different transformation techniques are used in DNA recombinant techniques. After successful insertion of the gene of interest into the selected vector, the researcher endeavours to choose an appropriate technique of incorporating the recombinant vector DNA into the cells of the chosen expression system. A classic example is the use of calcium chloride to increase the chances of bacterial cells absorbing a recombinant vector DNA. Different expression systems require varying transformational techniques. Culture of transformed expressions system Understanding the appropriate culture conditions of the chosen expression system is a determining factor of whether it will be possible to yield the heterologous protein of interest, this is because if the culture conditions prove unfavourable for the expression of system cells, minimal growth is registered reducing the chances of successful expression of the gene of interest. In any case, expression of the gene of interest occurs alongside the expression of other genes of the expression system cells. Evidently, all the expression systems described above require different culture conditions in a bid to increase the yield of the desired proteins; researchers have focused on understanding how to optimize the culture conditions for all the expression systems. However, such research ventures have only increased the understanding of maximizing yield in bacterial and yeast cells because of the salient understanding of optimal culture conditions in both of these expression systems (Lilie et al 2013, p. 1001). One of the critical factors in optimizing culture conditions is the appropriate choice of the culture medium, the amount of nutrients and substrates in the medium determine whether the cells have adequate nutrition to ensure fast growth. All the expression systems require different types of media a factor that compels a critical choice of the appropriate media to be made. Moreover, it is of significant importance for appropriate aeration to be allowed as it is a determining factor of growth. Research shows that the addition of an inducer of the production of the heterologous protein increases the chances of successful transcription the gene of interest yielding the protein. In addition, ensuring that the culture runs at the appropriate temperature is an essential step towards maximizing culture growth (Vinothkumar, Edwards, & Standfuss 2013, p 25). With the recent advances in understanding of the operation of different culture systems and practice, there is need to determine the best culture system for the production of the protein of interest. The choices to be made between batch cultures fed batch and continuous cultures. Each of these culture systems presents certain benefits and setbacks, therefore it is wise to choose the best depending on the protein of interest and the expression system. Notably, many industries that venture into large scale production of heterologous production or other bio products prefer the bath or fed batch culture system because of the minimal costs involve. Moreover, there is advanced understanding of these culture systems. However, continuing research on the efficiency of semi continuous and continuous cultures will serve to reduce the cost involved in these culture systems (Vinothkumar, Edwards, & Standfuss 2013, p 25). On the other hand, understanding of the semi continuous and the continuous cultures will serve to increase the automation of culture in sales. Diagram showing general steps in protein production Available at http://www.biosch.hku.hk/staff/mlc/mlc.html Factors affecting the expression of heterologous proteins : Expression system used Culture conditions Size of the desired heterologous proteins Transacting factors Cis acting factors Source of the heterologous proteins Degradation of the heterologous proteins Codon biasing Factors to consider when handling proteins It is critical to ensure that the activity and the stability of the heterologous protein is maintained throughout the process of production. This is because failure to maintain activity and stability yields proteins that have no use in the fields of interest. Moreover, it is important to ensure minimal chances of contamination in the entire procedure of heterologous protein production. Critical steps of ensuring that protein denaturation does not occur have to be taken if an active protein is to be yielded. Other issues requiring attention include proteolytic digestion, chemical modification, and adsorption (Ferrer-Miralle & Villaverde 2013, p. 10). Failure to keep these factors in consideration during the protein production compromise the quality and quantity of the heterologous protein produced. Factors to consider and the choice of method used in protein production It is important to maintain mild conditions in all the methods selected in the production of heterologous proteins in order to ensure that the produced proteins do not undergo denaturation. This is because proteins are sensitive to extreme conditions and may render them inactive after denaturation. An additional consideration is the concentration of solutions used in dire is different procedures (Vinothkumar, Edwards, & Standfuss 2013, p 25). There is a need to ensure that neither too low or too high concentrations are used as they affect protein activity. Moreover, it is wise to choose methods that ensure that the proteins produced are stable and do not undergo any form of degradation. The selected procedures should run at appropriate temperatures since high temperatures serve to denature proteins. The use of stabilizing agents such as glycerol increase the stability of the proteins produced. Depending on the protein of interest, it may prove inappropriate to use, reducing conditions or the freezer and thaw method (Krieger et al 2013, p. 500). Purification of heterologous proteins after production The method chosen for protein purification depends on the protein of interest and the expression system used. The purpose of purification is to get rid of any contaminants accumulated during different procedures in protein production. Moreover, it is critical to determine whether the protein is produced within the cell as a fusion protein or whether it is released into the medium. This is because proteins produced within the cell will require cell disruption as the initial step in the purification process. On the other hand, purification of proteins released into the medium begins immediately ((Vinothkumar, Edwards, & Standfuss 2013, p 23). Usually a tough protein is incorporated into the expression system in order to indicate whether the protein of interest has been produced. Moreover, the required purity of the proteins depends on the intended purpose for the generated protein. The activity of any protein depends on its purity and this place emphasis on the need to carry out effective purification. Research has revealed that there are several general considerations, in the purification process of proteins. One of the main objectives in the choice of the appropriate purification process seeks to maximize the yield of the purified proteins while minimizing the time and the cost involved. Since it is advisable to produce purified proteins, that still have their physiochemical attributes, it is important to select purifications methods that achieve this purpose. A main priority in protein purification is to achieve the removal of the major impurities in the initial steps. Experts advice that the most efficient method should be accorded priority while the method that involves higher costs should be a last option (Krieger et al 2013, p. 506). Different methods are of critical importance in the purification of proteins. With the factors highlighted above taken into consideration, selection of the most practical technique only serves to assure the biochemist of success in achieving the required level of purity of the desired protein. If the purification process is to be categorized as effective, the final product should contain only the desired type of molecule. In cases where proteins are produced within the cell, the most critical leading step is the facilitation of the release of the desired protein from the cell. This evidently serves as an initial step in the purification of fusion proteins. For this to occur, disruption of the cell membrane must be achieved. After the effective disruption of the cell, centrifugation follows. Centrifugation ensures successful separation of the supernatant. Repeated centrifugation that leads up to differential centrifugation can eventually release the desired protein from the cell (Krieger et al 2013, p. 502). After the release of proteins from the expression systems, the biochemist seeks to determine the order of events in the purification process. The order of procedures should present the capacity to yield a protein with high activity. The need to have a constant monitoring of the purification cannot receive any underestimation. Therefore, depending on the protein and purification, the biochemist should design an effective purification strategy. Methods of protein purification Salting out This is one of the techniques applicable in the purification of proteins. The basis of this technique is the use of varying salt concentrations in a bid to prompt the salting out process that ensures that concentration of the protein of interest. Dialysis Dialysis seeks to separate proteins through the use of a semipermeable membrane only some molecules whose size conforms to the permeability of the membrane can pass through, leaving large molecules under retention (Krieger et al 2013, p.503). Gel filtration This is an additional technique sued for the purification of proteins. Under this technique, loading of the desired sample onto a column of beads occurs after loading the smaller molecules find their way into the beads, while the larger molecules left at the top usually are the first to be separated (Cutler 2004, p.34). Gel filtration techniques are effective in separating proteins according to their sizes. Chromatography Evidently, chromatography is the commonest method of protein purification. In the initial phase, low-resolution chromatography yields a partially purified protein with a high resolution run of chromatography yielding the final pure protein. There are different types of chromatography techniques that are in use by many biochemists in a bid to diversify the techniques used in protein purification. Ion exchange chromatography is useful when charged proteins are under separation (Cutler 2004, p.2). In ion exchange chromatography, charge of the proteins determines its direction in the chromatography column. Affinity chromatography is an additional separation technique used in protein purification. This makes use of the capacity of different proteins to exhibit an affinity towards different types of proteins. In other cases, high pressure and reverse chromatography are of critical importance as preferred separation techniques. Gel electrophoresis This technique utilizes an electric current in a bid to separate proteins of different sizes and charges. After effective electrophoresis, the protein of interest separates from other molecules considered contaminants. Moreover, the use of isoelectric focusing is of critical importance as a separation technique after yielding certain proteins. The flow diagram indicates how certain techniques may follow each other in protein purification. Fermentation→ Sonication→ Membrane fractionation→ Heat treatment→ Ion-exchange chromatography→ Gel filtration. Membrane fractionation technique Sonication→ Centrifugation →Ultra centrifugation →washing → membrane washing using appropriate buffer →ultracentrifugation As the order above indicates, not all the separation techniques are critical in each step of the separation process. It is worth noting understanding the authors if the protein under production serves as an effective starting point that highlights the required procedures. In essence, different proteins are purified using a diverse range of techniques as the proteins they exhibit a salient diversity. The use of affinity tags in the process of separation of proteins is a critical step in ensuring that the produced protein has a tag that eases its identification. Failure to use the appropriate tags serves to complicate the purification process (Classen & Groth 2012, p. 130). One of the commonly used tags is the His tag, which has a remarkable recognition and is recommended because of its exhibited neutrality, and the fact that it does place the need for additional removal techniques. The inclusion of the tags depends on their attributes as well as those of the protein of interest occurring. In any case, there must be evidence of affinity that will facilitate the binding of the protein to the tag. Purification of Human PF4 protein from E. coli Evidently, researchers have described certain merits associated with the use of bacterial cells as preferred expression systems in some processes of protein production. With an increased understanding of gene expression in bacteria over the years, as well as the existing expertise of culturing such prokaryotic cells, there is a wide usage of bacterial cells in the production of heterologous recombinant cell. This has facilitated the use of Escherichia coli in the production of the human PF4 protein. An effective vector is critical as carrier of the gene of interest into the bacterial cell selected. With advanced understanding of the process if gene expression in E. coli, the recombinant DNA technology is easily achieved (Ichikawa 2013, p. 208). After successful transformation of E. coli cells using the calcium chloride, that prompts the intake of the transformed vector into the bacterial cells. Culturing of the transformed cells follows with the use of an effective selective media follows (Van Ooyen 2006, p. 381). Prior to the incubation of the transformed cells in the specialized medium used in the production of this protein, induction occurs. This is in accordance with research showing that the addition of an inducer of the production of the heterologous protein increases the chances of successful transcription the gene of interest yielding the protein. After the inclusion of the inducer, the human PF4 gene undergoes expression after an overnight incubation. After the successful expression of the human PF4 gene, the purification process follows. This method of production is a critical determinant of the separation technique adopted. The disruption of cells forms the initial step as it is an evident necessity since the human PF4 protein is yielded as a fusion protein (’ Protein production and purification’ 2008, p. 189). As highlighted above, the process of cell disruption involves several centrifugation runs, until efficient fractionation occurs. After the success of the above procedures, the biochemist endeavours to define an effective strategy of purification. The initial steps of the purification process involve the preparation, extraction and the clarification of the sample. A rigorous process of isolation, concentration, and stabilization follow (Kim et al 2013, p. 170). As experts have defined, bulk contaminants are removed, while polishing defines the final step in protein purification. Since all the phases described in this essay are used require a salient choice of the best techniques among the rest, the biochemist should have proper understanding of the protein, and the realization that proteins produced should exhibit a required level of activity (Ardisson-Araújo 2013, p. 162). This means that proteins should still have their physiochemical attributes, it is important to select purifications methods that achieve this purpose. Therefore, the defined priority in protein purification is to achieve the removal of the major impurities in the initial steps. According to advice from biochemists with expertise, the most efficient method should be accorded priority while the method that involves higher costs should be a last option. The polishing part involves the second phase of purification that relies in reverse chromatography or high-pressure chromatography (Bae et al 2013, p.8). Conclusion After understanding the protein of interest, it is critical to consider sequencing the gene coding for the protein. Evidently, without successful sequencing of the gene responsible for coding the protein, it is impossible to produce the desired protein. Moreover, heterologous proteins exhibit a remarkably high molecular weight, and require post-translational modifications and effective folding into active proteins (Pokoj et al 2009, 70). These processes are of critical significance as they determine the success of any industrial scale venture that aims at producing heterologous proteins. After production of the protein, purification is critical. BibliographyTop of Form ’ Protein production and purification’ 2008, Nat Methods, 5, 2, pp. 135–146. doi:10.1038/nmeth.f.202. Ardisson-Araújo, D, Rocha, J, da Costa, M, Bocca, A, Dusi, A, de Oliveira Resende, R, & Ribeiro, B 2013, A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification, Virology Journal, 10, p. 262, MEDLINE with Full Text, EBSCOhost, viewed 17 March 2014. 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