Biology论文模板 – Bioanalytical Technologies Application Report

Bioprocessing and DNA Analysis

a. Introduction

Proteins are the building blocks which are synthesised by all living forms through natural metabolism processes. Enzymes such as catalysts have a role in increasing the rate of metabolic reactions while others form cytoskeletons. Proteins play significant roles in various capacities in the body in aspects such as signalling immune responses, the cell cycle, and cell adhesion. Proteins can also be commercially produced in the industrial context using protein engineering and genetic engineering techniques. Both native and recombinant proteins play significant roles in sectors such as biopharmaceutical industry and agricultural industries. Products from these industries are widely applied in the fields of medicine, food and nutrition, polymers and plastics, detergent, textiles, pulp, paper and diagnostics (Demain & Vaishnav 2009).  The beginning of the twenty-first century saw more than 200 approved pharmaceutical products and vaccines having been developed by biopharmaceutical industries. Currently, there are over 200 approved peptide protein pharmaceutical products on the Food and Drug Administration List (Walsh 2014). This includes recombinant protein pharmaceutical products such Human Growth Hormone (HGH), insulin, Factor VIII, albumin and others. These pharmaceuticals have played significant roles in improving life in one way or another (Neidhardt 1996)

Recombinant DNA technology makes it possible for therapeutic proteins and protein molecules to be produced in large scale. Genetic modifications in proteins have the advantages over chemical modifications since those manufactured through the latter process are both biocompatible and biodegradable. This is because changes are introduced in a hundred percent of the molecules while excluding the rare errors in either gene transcription or translation. Further, the resulting proteins contain no residual amounts of undesired chemicals used during conjugation.

Bacterial expression systems are often preferred due to their simplicity and ability not to produce a recombinant human protein that is identical to naturally occurring wild type. Bacteria did not develop advanced ways for carrying out posttranslational modifications which do exist in higher organisms. As a result, there is an increment in the number of protein therapeutics expression in mammals. Nevertheless, the low costs and simple cultivation practices for bacteria give it unbeatable advantages over other forms of expression; thus, E. coli stands out as the most preferable choice on both a laboratory scale and in industrial scales (Kamionka 2011). Protein engineering can be used to overcome some of the posttranslational modifications in the bacterial cells of E. coli.

This report concentrates on the use of E. coli as a host for the engineering of insulin. E. coli is one of the earliest and widely adopted microbial choice of expression of most proteins (Chen 2012). Some of the advantages include rapid growth and expression, ease of culturing and the resulting high product yields (Neidhardt 1996). Thus, it is adopted for the massive production in large scale systems for the production of commercial proteins. Insulin is a notable in light of the fact that it was the earliest engineered protein and likewise an easy one to duplicate in the lab scale.

b. Experimental procedures

The simple illustration of the production of insulin using recombinant DNA technology follows the following process; Identification and removal of the human gene responsible for insulin production, opening of plasmid walls, followed by sealing the human gene into the plasmid using a ligase enzyme and then inserting the plasmid back into the bacterium, lastly, this is followed by growing of the plasmid into large numbers which all produce insulin. This can be illustrated using the following flow diagram:

(Lutz 2012)

In detail, the genetic engineering process for the manufacture of human insulin includes the following key steps:

  • Gene isolation

The gene that is responsible for the production of insulin is identified, isolated and removed using restriction enzymes such as EcoR1. Restriction proteins do not cut directly across the double strand of DNA since this may lead to a section of DNA being cut into several different pieces, hence making it impossible to isolate and entire gene. Instead, restriction proteins cut across the double strands at two different points. The cutting point is known as a sticky end. Restriction enzymes are used in cutting out specific genes, and also the cutting open places in the DNA plasmid for exact fitting of the genes (National Research Council (US) Committee on Bioprocess Engineering 1992).

(Sandhu 2010; Walsh 2013)

 After isolation, the gene may be copied in order to have several insulin genes to work with.

  • Preparation of target DNA

Using the same restriction protein technique, the plasmid wall of the bacterium is opened.

  • Inserting DNA into plasmid

With the plasmid ring open, the isolated human insulin gene is inserted into the plasmid ring and the ring closed. This is the actual recombination of the human gene with the bacterial plasmid. Sealing is done using a ligase enzyme.

Inserting the insulin gene into the plasmid

Inserting the plasmid back into the bacteria

  • Multiplication of plasmids

The plasmid containing the insulin genes are inserted into several bacterial cells. The cells are then fermented to grow. The fermentation process supplies the cells with nutrients necessary for them to grow, divide and survive. During their growth, the bacterial cell process will activate the gene for human insulin production and it will lead to the production of insulin in the cell. Reproduction of the cells through binary fission leads to division of the human insulin gene in the cells which are newly created (Endonucleases 2010; Johnson 1983).

Recombinant DNA put in large fermentation tanks

  • Reproduction in target cells

The human insulin protein molecules produced in the bacteria are then collected and purified

The bacterium uses the gene to produce insulin

Insulin is harvested from the bacteria

  • Purification

The substance is then purified for use as medicine.

Source: (Glick & Pasternak 2003; Brannigan & Wilkinson 2002)

In the lab, the steps that will be required to produce a recombinant E. coli colony expressing a recombinant protein, and completion of the desired post-biosynthesis, chemical transformations, and purification can be illustrated using the following process prescribes by the Center for Chemical Process Safety (CCPS).

The laboratory procedure will be translated to commercial production using the following steps

Source: (Center for Chemical ProcessSafety 2011)

Conclusion

Protein engineering is widely applied in the current world and it has been significantly highlighted in numerous studies and literature reviews. Applications for these engineering applications range from biocatalysts for the food industry to medical, nanobiotechnology and environmental applications. The use of bacteria, particularly E. coli expression system is the most preferred choice in the engineering of proteins.  It is several decades since insulin was manufactured as the first human medicine utilising recombinant DNA technology. The use of bacteria has advantages such as improved yield and ease of cultivation but equally has drawbacks such as the inability to carry out posttranslational modifications. Recombinant DNA technology is a significant biotechnology process.

References

Brannigan, J.A. & Wilkinson, A.J., 2002. Protein engineering 20 years on. Nature reviews. Molecular cell biology, 3(12), pp.964–70.

Center for Chemical ProcessSafety, 2011. Guidelines for Process Safety in Bioprocess Manufacturing Facilities, John Wiley & Sons.

Chen, R., 2012. Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnology Advances, 30(5), pp.1102–1107.

Demain, A.L. & Vaishnav, P., 2009. Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27(3), pp.297–306.

Endonucleases, R., 2010. Recombinant DNA. Calculations for Molecular Biology and Biotechnology, 7, pp.313–367.

Glick, B.R. & Pasternak, J.J., 2003. Molecular biotechnology: principles and applications of recombinant DNA,

Johnson, I.S., 1983. Human insulin from recombinant DNA technology. Science (New York, N.Y.), 219(4585), pp.632–637.

Kamionka, M., 2011. Engineering of therapeutic proteins production in Escherichia coli. Current pharmaceutical biotechnology, 12(2), pp.268–274.

Lutz, S., 2012. Protein Engineering Handbook, Volume 3, John Wiley & Sons.

National Research Council (US) Committee on Bioprocess Engineering, 1992. Current Bioprocess Technology, Products, and Opportunities.

Neidhardt, F.C., 1996. Escherichia Coli and Salmonella: Cellular and Molecular Biology, ASM Press.

Sandhu, S.S., 2010. Recombinant DNA Technology, I. K. International Pvt Ltd.

Walsh, G., 2014. Biopharmaceutical benchmarks 2014. Nature biotechnology, 32(7), pp.992–1000.

Walsh, G., 2013. Pharmaceutical Biotechnology: Concepts and Applications, John Wiley & Sons.

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