Intensification of inclusion body purification and protein refolding

There is an increasing need to translate genomic information into commercial products through protein expression and production. Formation of the protein as solid inclusion bodies in E. coli is advantageous as it enables good initial purification, provided that acceptable refolding yields can be ach...

Full description

Saved in:

Bibliographic Details
Main Author:	Lee, Chew Tin
Format:	Thesis
Language:	English
Published:	2003
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/6433/1/ChewTinLeePFKT2003.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-utm-ep.6433
record_format	uketd_dc
spelling	my-utm-ep.64332018-09-27T04:01:40Z Intensification of inclusion body purification and protein refolding 2003-08 Lee, Chew Tin TP Chemical technology There is an increasing need to translate genomic information into commercial products through protein expression and production. Formation of the protein as solid inclusion bodies in E. coli is advantageous as it enables good initial purification, provided that acceptable refolding yields can be achieved. However, the recovery of active recombinant protein from complex biological mixtures via the route of IB-formation involves a series of complicated recovery steps, each of which can compromise the purity and yield of the desired product. Purification of IBs using traditional methods of homogenisation and centrifugation are difficult to automate for high-throughput applications, and are costly to scale. Process intensification by minimising the number of unit operations without a loss of product purity is desirable to improve yield and reduce production cost. Such a process should ideally be generic, scalable, easily automated to facilitate continuous processing, and should result in an improved refolding yield. Based on these criteria, an intensified unit operation termed an integrated Membrane Oscillatory Refolding (MOR) is introduced. The integrated MOR unit consists of an oscillatory flow reactor (OFR) that is integrated with a hollow ceramic membrane for cross-flow microfiltration operation. The key is the use of innovative chemical extraction technology in a non-solubilising mode for IB release, coupled with the MOR unit that acts as a novel microfiltration unit for IB purification and protein refolding. The technologies rely only on chemical reagents and on microfiltration that can be easily scaled and automated. The objective of this study is to establish and research the three key process steps essential to realise the integrated MOR unit: a non-solubilising chemical extraction method; a cross-flow microfiltration for IB recovery; and protein refolding via the hollow ceramic membrane in a novel reactor. Three of these process steps, validated using granulocyte macrophage-colony stimulating factor (GM-CSF) IBs, are successfully demonstrated and ready for integration into a single unit of MOR 2003-08 Thesis http://eprints.utm.my/id/eprint/6433/ http://eprints.utm.my/id/eprint/6433/1/ChewTinLeePFKT2003.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:62270 phd doctoral University of Cambridge, London, Churchill College, Department of Chemical Engineering Churchill College, Department of Chemical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Lee, Chew Tin Intensification of inclusion body purification and protein refolding
description	There is an increasing need to translate genomic information into commercial products through protein expression and production. Formation of the protein as solid inclusion bodies in E. coli is advantageous as it enables good initial purification, provided that acceptable refolding yields can be achieved. However, the recovery of active recombinant protein from complex biological mixtures via the route of IB-formation involves a series of complicated recovery steps, each of which can compromise the purity and yield of the desired product. Purification of IBs using traditional methods of homogenisation and centrifugation are difficult to automate for high-throughput applications, and are costly to scale. Process intensification by minimising the number of unit operations without a loss of product purity is desirable to improve yield and reduce production cost. Such a process should ideally be generic, scalable, easily automated to facilitate continuous processing, and should result in an improved refolding yield. Based on these criteria, an intensified unit operation termed an integrated Membrane Oscillatory Refolding (MOR) is introduced. The integrated MOR unit consists of an oscillatory flow reactor (OFR) that is integrated with a hollow ceramic membrane for cross-flow microfiltration operation. The key is the use of innovative chemical extraction technology in a non-solubilising mode for IB release, coupled with the MOR unit that acts as a novel microfiltration unit for IB purification and protein refolding. The technologies rely only on chemical reagents and on microfiltration that can be easily scaled and automated. The objective of this study is to establish and research the three key process steps essential to realise the integrated MOR unit: a non-solubilising chemical extraction method; a cross-flow microfiltration for IB recovery; and protein refolding via the hollow ceramic membrane in a novel reactor. Three of these process steps, validated using granulocyte macrophage-colony stimulating factor (GM-CSF) IBs, are successfully demonstrated and ready for integration into a single unit of MOR
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Lee, Chew Tin
author_facet	Lee, Chew Tin
author_sort	Lee, Chew Tin
title	Intensification of inclusion body purification and protein refolding
title_short	Intensification of inclusion body purification and protein refolding
title_full	Intensification of inclusion body purification and protein refolding
title_fullStr	Intensification of inclusion body purification and protein refolding
title_full_unstemmed	Intensification of inclusion body purification and protein refolding
title_sort	intensification of inclusion body purification and protein refolding
granting_institution	University of Cambridge, London, Churchill College, Department of Chemical Engineering
granting_department	Churchill College, Department of Chemical Engineering
publishDate	2003
url	http://eprints.utm.my/id/eprint/6433/1/ChewTinLeePFKT2003.pdf
_version_	1747814658675310592

Intensification of inclusion body purification and protein refolding

Similar Items