Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions

Human epidermal growth factor (hEGF) is a 6.2 kD polypeptide secreted by human tissues into the body fluid. It carries multiple biological functions including induction of tissue proliferation and differentiation, a process that is important in tissue repair. Because of this function, hEGF is widely...

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Main Author:	Eissazadeh, Samira
Format:	Thesis
Language:	English
Published:	2012
Subjects:	Epidermal growth factor Pichia pastoris Fermentation
Online Access:	http://psasir.upm.edu.my/id/eprint/31605/1/FBSB%202012%2030R.pdf
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spelling	my-upm-ir.316052015-02-02T07:44:03Z Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions 2012-07 Eissazadeh, Samira Human epidermal growth factor (hEGF) is a 6.2 kD polypeptide secreted by human tissues into the body fluid. It carries multiple biological functions including induction of tissue proliferation and differentiation, a process that is important in tissue repair. Because of this function, hEGF is widely used in various medical and industrial applications such as for wound healing, corneal transplant and treatment of gastric ulcers. As a result, the demand for hEGF supply for local consumption is always high, and there is a need for enough and cheap local supply of this protein. However, local production of hEGF especially in large commercial scales has not been successful due to various technical challenges. This study was carried out to express the hEGF gene in the yeast, Pichia pastoris GS115 using a commercially available expression vector equipped with a specific signal peptide that directs the transportation of the hEGF protein into the culture medium. The performance of the expression system for hEGF production was enhanced by optimizing the fermentation conditions of the yeast. The hEGF gene was ligated into the pPIC9K expression vector, and then integrated into the yeast’s genome by electroporation. The hEGF was assayed by using an ELISA-based system and the recombinant cell line that produced the highest hEGF was then used in the optimization of the fermentation conditions. In addition, the amount of methanol, an inducer for the expression system was also optimised. The highest amount of hEGF secreted into the medium was obtained from clone B1 of the recombinant yeast with an average yield of 2.27 μg/ml. This was obtained through an induction of the yeast culture with 0.5% (v/v) methanol for 60 hours. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. Within this pH range, the difference in hEGF production was not significant. Two fermentation conditions commonly known to affect heterologous protein production (pH and temperature) were analyzed by using the artificial neural network (ANN). Changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature. As anticipated from the model, the production of hEGF was decreased when the pH of the culture medium was lower than 6.0, or when the temperature was lower than 30°C. As a conclusion, hEGF was correctly secreted into the culture media of the recombinant P. pastoris as anticipated from the properties of the expression vector used. The amount of hEGF secreted into the medium was affected by the fermentation conditions especially the types of culture media used, the pH of the culture media and incubation temperature. Epidermal growth factor Pichia pastoris Fermentation 2012-07 Thesis http://psasir.upm.edu.my/id/eprint/31605/ http://psasir.upm.edu.my/id/eprint/31605/1/FBSB%202012%2030R.pdf application/pdf en public masters Universiti Putra Malaysia Epidermal growth factor Pichia pastoris Fermentation Faculty of Biotechnology and Biomolecular Sciences
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Epidermal growth factor Pichia pastoris Fermentation
spellingShingle	Epidermal growth factor Pichia pastoris Fermentation Eissazadeh, Samira Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
description	Human epidermal growth factor (hEGF) is a 6.2 kD polypeptide secreted by human tissues into the body fluid. It carries multiple biological functions including induction of tissue proliferation and differentiation, a process that is important in tissue repair. Because of this function, hEGF is widely used in various medical and industrial applications such as for wound healing, corneal transplant and treatment of gastric ulcers. As a result, the demand for hEGF supply for local consumption is always high, and there is a need for enough and cheap local supply of this protein. However, local production of hEGF especially in large commercial scales has not been successful due to various technical challenges. This study was carried out to express the hEGF gene in the yeast, Pichia pastoris GS115 using a commercially available expression vector equipped with a specific signal peptide that directs the transportation of the hEGF protein into the culture medium. The performance of the expression system for hEGF production was enhanced by optimizing the fermentation conditions of the yeast. The hEGF gene was ligated into the pPIC9K expression vector, and then integrated into the yeast’s genome by electroporation. The hEGF was assayed by using an ELISA-based system and the recombinant cell line that produced the highest hEGF was then used in the optimization of the fermentation conditions. In addition, the amount of methanol, an inducer for the expression system was also optimised. The highest amount of hEGF secreted into the medium was obtained from clone B1 of the recombinant yeast with an average yield of 2.27 μg/ml. This was obtained through an induction of the yeast culture with 0.5% (v/v) methanol for 60 hours. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. Within this pH range, the difference in hEGF production was not significant. Two fermentation conditions commonly known to affect heterologous protein production (pH and temperature) were analyzed by using the artificial neural network (ANN). Changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature. As anticipated from the model, the production of hEGF was decreased when the pH of the culture medium was lower than 6.0, or when the temperature was lower than 30°C. As a conclusion, hEGF was correctly secreted into the culture media of the recombinant P. pastoris as anticipated from the properties of the expression vector used. The amount of hEGF secreted into the medium was affected by the fermentation conditions especially the types of culture media used, the pH of the culture media and incubation temperature.
format	Thesis
qualification_level	Master's degree
author	Eissazadeh, Samira
author_facet	Eissazadeh, Samira
author_sort	Eissazadeh, Samira
title	Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
title_short	Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
title_full	Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
title_fullStr	Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
title_full_unstemmed	Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
title_sort	expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2012
url	http://psasir.upm.edu.my/id/eprint/31605/1/FBSB%202012%2030R.pdf
_version_	1747811617941225472

Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions

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