Characterisation of a novel stimuli-responsive polymers for protein refolding

During bioprocessing, there are many points at which protein can aggregate, reducing the overall protein refolding yield. A novel stimuli responsive polymer: Poly (L-lysine iso-phthalamide) was investigated in this study to examine its potential use as an additive to improve refolding yield of hen e...

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Main Author: Ang, Jing Mei
Other Authors: Susanna Leong Su Jan
Format: Final Year Project
Language:English
Published: 2009
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Online Access:http://hdl.handle.net/10356/16651
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-166512023-03-03T15:32:05Z Characterisation of a novel stimuli-responsive polymers for protein refolding Ang, Jing Mei Susanna Leong Su Jan School of Chemical and Biomedical Engineering DRNTU::Engineering::Chemical engineering::Biotechnology During bioprocessing, there are many points at which protein can aggregate, reducing the overall protein refolding yield. A novel stimuli responsive polymer: Poly (L-lysine iso-phthalamide) was investigated in this study to examine its potential use as an additive to improve refolding yield of hen egg white lysozyme (HEWL). Poly (L-lysine iso-phthalamide) is anionic at pH 6.0 or higher and cationic when the environmental pH falls below pH 4.0. Phase transition of the polymer from soluble state to an insoluble state occurs between pH 4.0 to 6.0. Investigations were then carried out to determine the optimal conditions to carry out lysozyme refolding. Factors that influence lysozyme refolding yield such as (i) protein refolding pH, (ii) polymer concentration in refolding buffer and (iii) urea concentration in refolding buffer were examined. Maximum lysozyme refolding yield of 22% was achieved when refolding was carried out using a refolding buffer consisted of 0.2 W/V% Poly (L-lysine iso-phthalamide), 2.0M urea and 4mM EDTA at pH 8. A control study was also carried out to investigate the effects of GSH and GSSG on lysozyme refolding yield. GSH and GSSG are redox agents that are normally added into refolding buffer to improve protein refolding yield. The addition of GSH and GSSG helps improve lysozyme refolding yield as they promote disulfide bond formation. However, Poly (L-lysine iso-phthalamide) was found to be ineffective in improving lysozyme refolding yield when GSH and GSSG were added into the refolding buffer. In fact, 68% lysozyme refolding yield was achieved when no polymer was used in the presence of GSH and GSSG. When the polymer is added into this refolding buffer, lysozyme refolding yield was reduced to less than 10%. This finding suggests that possible molecular interactions between the polymer, GSH and GSSG are inhibiting lysozyme refolding hence the decrement in refolding yield. Bachelor of Engineering (Chemical and Biomolecular Engineering) 2009-05-27T08:48:29Z 2009-05-27T08:48:29Z 2009 2009 Final Year Project (FYP) http://hdl.handle.net/10356/16651 en Nanyang Technological University 58 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Engineering::Chemical engineering::Biotechnology
spellingShingle DRNTU::Engineering::Chemical engineering::Biotechnology
Ang, Jing Mei
Characterisation of a novel stimuli-responsive polymers for protein refolding
description During bioprocessing, there are many points at which protein can aggregate, reducing the overall protein refolding yield. A novel stimuli responsive polymer: Poly (L-lysine iso-phthalamide) was investigated in this study to examine its potential use as an additive to improve refolding yield of hen egg white lysozyme (HEWL). Poly (L-lysine iso-phthalamide) is anionic at pH 6.0 or higher and cationic when the environmental pH falls below pH 4.0. Phase transition of the polymer from soluble state to an insoluble state occurs between pH 4.0 to 6.0. Investigations were then carried out to determine the optimal conditions to carry out lysozyme refolding. Factors that influence lysozyme refolding yield such as (i) protein refolding pH, (ii) polymer concentration in refolding buffer and (iii) urea concentration in refolding buffer were examined. Maximum lysozyme refolding yield of 22% was achieved when refolding was carried out using a refolding buffer consisted of 0.2 W/V% Poly (L-lysine iso-phthalamide), 2.0M urea and 4mM EDTA at pH 8. A control study was also carried out to investigate the effects of GSH and GSSG on lysozyme refolding yield. GSH and GSSG are redox agents that are normally added into refolding buffer to improve protein refolding yield. The addition of GSH and GSSG helps improve lysozyme refolding yield as they promote disulfide bond formation. However, Poly (L-lysine iso-phthalamide) was found to be ineffective in improving lysozyme refolding yield when GSH and GSSG were added into the refolding buffer. In fact, 68% lysozyme refolding yield was achieved when no polymer was used in the presence of GSH and GSSG. When the polymer is added into this refolding buffer, lysozyme refolding yield was reduced to less than 10%. This finding suggests that possible molecular interactions between the polymer, GSH and GSSG are inhibiting lysozyme refolding hence the decrement in refolding yield.
author2 Susanna Leong Su Jan
author_facet Susanna Leong Su Jan
Ang, Jing Mei
format Final Year Project
author Ang, Jing Mei
author_sort Ang, Jing Mei
title Characterisation of a novel stimuli-responsive polymers for protein refolding
title_short Characterisation of a novel stimuli-responsive polymers for protein refolding
title_full Characterisation of a novel stimuli-responsive polymers for protein refolding
title_fullStr Characterisation of a novel stimuli-responsive polymers for protein refolding
title_full_unstemmed Characterisation of a novel stimuli-responsive polymers for protein refolding
title_sort characterisation of a novel stimuli-responsive polymers for protein refolding
publishDate 2009
url http://hdl.handle.net/10356/16651
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