Therapeutic Proteins – Hirudin

1436 words (6 pages) Essay

29th Jan 2018 Chemistry Reference this

Disclaimer: This work has been submitted by a university student. This is not an example of the work produced by our Essay Writing Service. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UKEssays.com.

Isaac Johnson

Hirudin is a polypeptide that has anticoagulant properties (Rosenthal, 2008). Hirudin is important because it is a direct thrombin inhibitor. Since the presence of thrombin, a serine protease, is indicative of a blood clot having a polypeptide that specifically inhibits thrombin generation is important in a number of ways (Contributors, Thrombin, 2013). Hirudin is used to prevent the formation of blood clots in arteries, veins, and chambers within the heart (Rosenthal, 2008). Hirudins produced by recombinant biotechnology are more effective than other serine proteases produced by the body because they break up several different types of thrombin and they don’t bind to plasma proteins, which means there is a known dose-response relationship (Andreas Greinacher, 2008). The clinical use of r-hirudin provided an alternative choice to those people who experienced complications when given heparin; in addition “r-hirudin is important because it binds directly to thrombin, thereby forming a complex with thrombin not only at its fibrinogen-binding site but also at its catalytic region”(Eichinger, 1995, p. 886), which completely stops the generation of thrombin unlike heparin which stops the production of thrombin by “catalyzing the inactivation of thrombin using anti-thrombin III”(Eichinerger, 1995, p. 886).

Get Help With Your Essay

If you need assistance with writing your essay, our professional essay writing service is here to help!

Find out more

Hirudin was discovered by John Haycraft in 1884 during his research on the coagulation of blood. In the 1900s it was extracted from leeches but today it is produced through recombinant biotechnology (Contributors, Hirudin, 2014). “The first recombinant hirudin produced and approved for clinical use, called lepirudin, was approved by the FDA in 1998 for the treatment of heparin-induced thrombocytopenia complicated by thrombosis” (Greinacher, 2008, p. 819). Heparin-induced thrombocytopenia is a disorder which causes clotting to occur instead of excessive bleeding and is countered by administering another anticoagulant to inhibit thrombocytosis (Contributors, Heparin-induced thrombocytopenia, 2013).

Hirudin is a polypeptide produced by the parapharyngeal glands of the leech, Hirudo medicinalis. Hirudins produced through recombinant biotechnology are generally produced using Pichia pastorus although Escherichia coli can be used as well albeit with the risk of degradation (Wuguang Lu, 2012). The process of producing recombinant hirudin (rHIR) is a multi-stage process, the main stages are: Construction of HIR/pPic9k, Transformation of P. pastorus strain, Shake flask evaluation of transformants, Clone fermentation, rHIR purification (Stuart A. Rosenfeld, 1996).

The first stage, the construction of HIR/pPic9k, had several steps: use of pLMFα1 as a template for the PCR amplication of the hirudin gene, the use of primers to clone the gene, and the use of restrictive endonucleases to cut out part of the vector so that the hirudin gene could be inserted into pPic9K vector. Because the hirudin gene was inserted into the expression vector it allowed for greater control of the cloning process and allowed for optimization of the process. In the second stage, the transformation of P. pastorus strain, the expression vector was digested with Sa1I, a restrictive endonuclease, and the DNA was integrated into the host genome through homologous recombination via electroporation. Because the Hir/PPic9K was digested with Sa1I a large amount of His+ transformants were obtained after electroporation. These transformants were replica-plated, in order to obtain resistance to the antibiotic G418, whereupon the transformants could then undergo multiple chromosomal integrations of the HIR gene. In the third stage, the shake flask evaluation of transformants, the transformants were screened to determine if they were capable of expressing and secreting recombinant hirudin. All of the transformants from the experiment did secrete recombinant hirudin albeit at different rates over the course of the 7 day evaluation. In the fourth stage, clone fermentation, the cultures were fermented in order to increase production of recombinant hirudin. Fermentation conditions increased r-hir production based on dissolved oxygen content, nutrient balance and high cell densities which made the environment optimal for recombinant hirudin production. The fermentation process was a batch process and used glycerol as the sole carbon source followed later by feeding the cultures methanol to fully induce rHIR expression. The methanol feed rate was varied so that a low concentration could be maintained in the fermentor thus keeping toxicity levels low. A low pH level is required for the fermentation process; a pH of 5 is too high and causes the proteins to break down while a pH of 3 is optimal for rHIR production. The final stage, the purification of rHIR, used a two-step purification protocol where first Q-Sepharose ion exchange chromatography was used followed by preparative high-performance liquid chromatography (HPLC). The recombinant hirudin had a purity percentage of more than 97% and a recovery yield of 63% (Stuart A. Rosenfeld, 1996).

The impact of therapeutic proteins on society has been overwhelmingly positive. Recombinant proteins have many uses in society and have been incorporated into many different facets of everyday life. They are used in many different types of therapy from growth hormone treatment in adults to the use of recombinant interferon used to treat Hepatitis C. Recombinant proteins don’t just impact the medical field though they also play an important role in cosmetics and as dietary supplements. Furthermore when recombinant proteins are compared to blood plasma-derived products they are clearly shown to be safer. Since recombinant proteins were first introduced in the early 1990s there have been few adverse effects caused by them and zero fatalities whereas blood plasma-derived products carry the risk of transmission of emerging diseases that could turn into epidemics like the swine flu or bird flu (Liras, 2008).

In conclusion hirudin is a polypeptide that is a direct inhibitor of thrombin and thus is an excellent anticoagulant. Several different types of recombinant proteins derived from hirudin have been approved for clinical use since the late 1990’s. These alternatives, like lepirudin, fill a necessary niche in the medical world that other coagulants cannot fill due to either patient complications with those drugs or hirudin derived drugs simply being more effective. The role that therapeutic proteins play in medical treatments and therapy will increase in society at they become more cost effective and production methods become more efficient.

References

Andreas Greinacher, T. E. (2008). The direct thrombin inhibitor hirudin . Thromb Haemost, 819-829.

Contributors, W. (2013, November 2). Heparin-induced thrombocytopenia. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Heparin-induced_thrombocytopenia

Contributors, W. (2013, December 23). Thrombin. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Thrombin

Contributors, W. (2014, January 4). Hirudin. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Hirudin

Liras, A. (2008). Recombinant proteins in therapeutics: haemophilia treatment as an example. International Archives of Medicine, 1-4.

Rosenthal, S. O. (2008). Encylopedia of Molecular Pharmacology. New York: Springer-Verlag Berlin Heidelberg.

Sabine Eichinger, M. W.-L.-G. (1995). Effects of Recombinant Hirudin (r-Hirudin, HBW 023) on Coagulation and Platelet Activation In Vivo. Arteriosclerosis, Thrombosis, and Vascular Biology, 886-892.

Stuart A. Rosenfeld, D. N. (1996). Production and Purification of Recombinant Hirudin. PROTEIN EXPRESSION AND PURIFICATION Article No. 0127, 476-482.

Wuguang Lu, X. C. (2012). Production and Characterization of Hirudin Variant-1 by SUMO. Springer Science+Business Media, 41-48.

Isaac Johnson

Hirudin is a polypeptide that has anticoagulant properties (Rosenthal, 2008). Hirudin is important because it is a direct thrombin inhibitor. Since the presence of thrombin, a serine protease, is indicative of a blood clot having a polypeptide that specifically inhibits thrombin generation is important in a number of ways (Contributors, Thrombin, 2013). Hirudin is used to prevent the formation of blood clots in arteries, veins, and chambers within the heart (Rosenthal, 2008). Hirudins produced by recombinant biotechnology are more effective than other serine proteases produced by the body because they break up several different types of thrombin and they don’t bind to plasma proteins, which means there is a known dose-response relationship (Andreas Greinacher, 2008). The clinical use of r-hirudin provided an alternative choice to those people who experienced complications when given heparin; in addition “r-hirudin is important because it binds directly to thrombin, thereby forming a complex with thrombin not only at its fibrinogen-binding site but also at its catalytic region”(Eichinger, 1995, p. 886), which completely stops the generation of thrombin unlike heparin which stops the production of thrombin by “catalyzing the inactivation of thrombin using anti-thrombin III”(Eichinerger, 1995, p. 886).

Hirudin was discovered by John Haycraft in 1884 during his research on the coagulation of blood. In the 1900s it was extracted from leeches but today it is produced through recombinant biotechnology (Contributors, Hirudin, 2014). “The first recombinant hirudin produced and approved for clinical use, called lepirudin, was approved by the FDA in 1998 for the treatment of heparin-induced thrombocytopenia complicated by thrombosis” (Greinacher, 2008, p. 819). Heparin-induced thrombocytopenia is a disorder which causes clotting to occur instead of excessive bleeding and is countered by administering another anticoagulant to inhibit thrombocytosis (Contributors, Heparin-induced thrombocytopenia, 2013).

Hirudin is a polypeptide produced by the parapharyngeal glands of the leech, Hirudo medicinalis. Hirudins produced through recombinant biotechnology are generally produced using Pichia pastorus although Escherichia coli can be used as well albeit with the risk of degradation (Wuguang Lu, 2012). The process of producing recombinant hirudin (rHIR) is a multi-stage process, the main stages are: Construction of HIR/pPic9k, Transformation of P. pastorus strain, Shake flask evaluation of transformants, Clone fermentation, rHIR purification (Stuart A. Rosenfeld, 1996).

The first stage, the construction of HIR/pPic9k, had several steps: use of pLMFα1 as a template for the PCR amplication of the hirudin gene, the use of primers to clone the gene, and the use of restrictive endonucleases to cut out part of the vector so that the hirudin gene could be inserted into pPic9K vector. Because the hirudin gene was inserted into the expression vector it allowed for greater control of the cloning process and allowed for optimization of the process. In the second stage, the transformation of P. pastorus strain, the expression vector was digested with Sa1I, a restrictive endonuclease, and the DNA was integrated into the host genome through homologous recombination via electroporation. Because the Hir/PPic9K was digested with Sa1I a large amount of His+ transformants were obtained after electroporation. These transformants were replica-plated, in order to obtain resistance to the antibiotic G418, whereupon the transformants could then undergo multiple chromosomal integrations of the HIR gene. In the third stage, the shake flask evaluation of transformants, the transformants were screened to determine if they were capable of expressing and secreting recombinant hirudin. All of the transformants from the experiment did secrete recombinant hirudin albeit at different rates over the course of the 7 day evaluation. In the fourth stage, clone fermentation, the cultures were fermented in order to increase production of recombinant hirudin. Fermentation conditions increased r-hir production based on dissolved oxygen content, nutrient balance and high cell densities which made the environment optimal for recombinant hirudin production. The fermentation process was a batch process and used glycerol as the sole carbon source followed later by feeding the cultures methanol to fully induce rHIR expression. The methanol feed rate was varied so that a low concentration could be maintained in the fermentor thus keeping toxicity levels low. A low pH level is required for the fermentation process; a pH of 5 is too high and causes the proteins to break down while a pH of 3 is optimal for rHIR production. The final stage, the purification of rHIR, used a two-step purification protocol where first Q-Sepharose ion exchange chromatography was used followed by preparative high-performance liquid chromatography (HPLC). The recombinant hirudin had a purity percentage of more than 97% and a recovery yield of 63% (Stuart A. Rosenfeld, 1996).

The impact of therapeutic proteins on society has been overwhelmingly positive. Recombinant proteins have many uses in society and have been incorporated into many different facets of everyday life. They are used in many different types of therapy from growth hormone treatment in adults to the use of recombinant interferon used to treat Hepatitis C. Recombinant proteins don’t just impact the medical field though they also play an important role in cosmetics and as dietary supplements. Furthermore when recombinant proteins are compared to blood plasma-derived products they are clearly shown to be safer. Since recombinant proteins were first introduced in the early 1990s there have been few adverse effects caused by them and zero fatalities whereas blood plasma-derived products carry the risk of transmission of emerging diseases that could turn into epidemics like the swine flu or bird flu (Liras, 2008).

In conclusion hirudin is a polypeptide that is a direct inhibitor of thrombin and thus is an excellent anticoagulant. Several different types of recombinant proteins derived from hirudin have been approved for clinical use since the late 1990’s. These alternatives, like lepirudin, fill a necessary niche in the medical world that other coagulants cannot fill due to either patient complications with those drugs or hirudin derived drugs simply being more effective. The role that therapeutic proteins play in medical treatments and therapy will increase in society at they become more cost effective and production methods become more efficient.

References

Andreas Greinacher, T. E. (2008). The direct thrombin inhibitor hirudin . Thromb Haemost, 819-829.

Contributors, W. (2013, November 2). Heparin-induced thrombocytopenia. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Heparin-induced_thrombocytopenia

Contributors, W. (2013, December 23). Thrombin. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Thrombin

Contributors, W. (2014, January 4). Hirudin. Retrieved January 11, 2014, from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/wiki/Hirudin

Liras, A. (2008). Recombinant proteins in therapeutics: haemophilia treatment as an example. International Archives of Medicine, 1-4.

Rosenthal, S. O. (2008). Encylopedia of Molecular Pharmacology. New York: Springer-Verlag Berlin Heidelberg.

Sabine Eichinger, M. W.-L.-G. (1995). Effects of Recombinant Hirudin (r-Hirudin, HBW 023) on Coagulation and Platelet Activation In Vivo. Arteriosclerosis, Thrombosis, and Vascular Biology, 886-892.

Stuart A. Rosenfeld, D. N. (1996). Production and Purification of Recombinant Hirudin. PROTEIN EXPRESSION AND PURIFICATION Article No. 0127, 476-482.

Wuguang Lu, X. C. (2012). Production and Characterization of Hirudin Variant-1 by SUMO. Springer Science+Business Media, 41-48.

Cite This Work

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Related Services

View all

DMCA / Removal Request

If you are the original writer of this essay and no longer wish to have your work published on the UKDiss.com website then please: