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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...

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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
http://eprints.utm.my/id/eprint/6433/
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spelling my.utm.64332018-09-27T04:01:40Z http://eprints.utm.my/id/eprint/6433/ Intensification of inclusion body purification and protein refolding 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 NonPeerReviewed application/pdf en http://eprints.utm.my/id/eprint/6433/1/ChewTinLeePFKT2003.pdf Lee, Chew Tin (2003) Intensification of inclusion body purification and protein refolding. PhD thesis, University of Cambridge, London, Churchill College, Department of Chemical Engineering. http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:62270
institution Universiti Teknologi Malaysia
building UTM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Teknologi Malaysia
content_source UTM Institutional Repository
url_provider http://eprints.utm.my/
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
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
publishDate 2003
url http://eprints.utm.my/id/eprint/6433/1/ChewTinLeePFKT2003.pdf
http://eprints.utm.my/id/eprint/6433/
http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:62270
_version_ 1643644556907905024
score 13.211869

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