Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli

Because of its many beneficial features, Escherichia coli is widely used as host for recombinant protein production. However, one major disadvantage of using E. coli is the formation of inclusion body especially when the transgene is over-expressed. Therefore, an understanding of biochemical mechani...

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Main Author:	Heidary, Somayyeh
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Escherichia coli Proteins Recombinant proteins
Online Access:	http://psasir.upm.edu.my/id/eprint/38910/1/FBSB%202013%206.pdf
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id	my-upm-ir.38910
record_format	uketd_dc
spelling	my-upm-ir.389102024-08-29T04:40:50Z Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli 2013-01 Heidary, Somayyeh Because of its many beneficial features, Escherichia coli is widely used as host for recombinant protein production. However, one major disadvantage of using E. coli is the formation of inclusion body especially when the transgene is over-expressed. Therefore, an understanding of biochemical mechanism that triggers the formation of recombinant protein as inclusion bodies is important before the problem can be solved. To study the biochemical changes following an over-expression of a transgene in E. coli at protein level, differential periplasmic proteome was analyzed by using a two-dimensional gel electrophoresis technique. The recombinant E. coli RG 2(DE3) carrying the plasmid pET-26b that encodes a human interferon-α2b was used as a model organism. Crude protein extracts were prepared from the periplasmic space of the E. coli cells by using an osmotic shock method. The protein samples were then separated on a 2D gel. High resolution of protein spots were successfully obtained from the protein samples after some optimizations were done on the rehydration buffer components. Optimization of CHAPS, ampholyte and DTT concentration and isoelectric focusing procedure had most effects on 2D result. Based on the software analysis of the protein spots obtained, some potential unique, up- and down-regulated protein spots were observed. Most of the up and down regulated identified proteins were shown to be involved in ABC-transporter protein family such as phosphate ABC transporter, glutathione ABC transporter and oligopeptide ABC transporter. Knowing the types of protein family responded to the transgene over-expression may provide an important clue to what triggers E.coli to produce inclusion bodies. Escherichia coli Proteins Recombinant proteins 2013-01 Thesis http://psasir.upm.edu.my/id/eprint/38910/ http://psasir.upm.edu.my/id/eprint/38910/1/FBSB%202013%206.pdf text en public masters Universiti Putra Malaysia Escherichia coli Proteins Recombinant proteins Abdullah, Mohd Puad
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Abdullah, Mohd Puad
topic	Escherichia coli Proteins Recombinant proteins
spellingShingle	Escherichia coli Proteins Recombinant proteins Heidary, Somayyeh Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
description	Because of its many beneficial features, Escherichia coli is widely used as host for recombinant protein production. However, one major disadvantage of using E. coli is the formation of inclusion body especially when the transgene is over-expressed. Therefore, an understanding of biochemical mechanism that triggers the formation of recombinant protein as inclusion bodies is important before the problem can be solved. To study the biochemical changes following an over-expression of a transgene in E. coli at protein level, differential periplasmic proteome was analyzed by using a two-dimensional gel electrophoresis technique. The recombinant E. coli RG 2(DE3) carrying the plasmid pET-26b that encodes a human interferon-α2b was used as a model organism. Crude protein extracts were prepared from the periplasmic space of the E. coli cells by using an osmotic shock method. The protein samples were then separated on a 2D gel. High resolution of protein spots were successfully obtained from the protein samples after some optimizations were done on the rehydration buffer components. Optimization of CHAPS, ampholyte and DTT concentration and isoelectric focusing procedure had most effects on 2D result. Based on the software analysis of the protein spots obtained, some potential unique, up- and down-regulated protein spots were observed. Most of the up and down regulated identified proteins were shown to be involved in ABC-transporter protein family such as phosphate ABC transporter, glutathione ABC transporter and oligopeptide ABC transporter. Knowing the types of protein family responded to the transgene over-expression may provide an important clue to what triggers E.coli to produce inclusion bodies.
format	Thesis
qualification_level	Master's degree
author	Heidary, Somayyeh
author_facet	Heidary, Somayyeh
author_sort	Heidary, Somayyeh
title	Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
title_short	Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
title_full	Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
title_fullStr	Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
title_full_unstemmed	Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
title_sort	periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli
granting_institution	Universiti Putra Malaysia
publishDate	2013
url	http://psasir.upm.edu.my/id/eprint/38910/1/FBSB%202013%206.pdf
_version_	1811767730264604672

Periplasmic protein changes in response to over-expression of recombinant protein in escherichia coli

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