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Optimising the refolding conditions of a recombinant protein. / Duha Ghaboush Eldawi

Swansea University Author: Duha Ghaboush Eldawi

Abstract

The objective of this project is to increase the yield and the quality of in vitro refolded proteins over-expressed in E. coli as insoluble inclusion bodies by optimising the refolding conditions. Therefore, protein refolding additives and regimes like (Glycerol, Gnd-HCl, Dilution, Temperature, and...

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Published: 2008
Institution: Swansea University
Degree level: Master of Philosophy
Degree name: M.Phil
URI: https://cronfa.swan.ac.uk/Record/cronfa42474
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first_indexed 2018-08-02T18:54:47Z
last_indexed 2019-10-21T16:47:53Z
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spelling 2018-08-16T14:39:02.9105634 v2 42474 2018-08-02 Optimising the refolding conditions of a recombinant protein. e8b3692eae92308877907a67dc883673 NULL Duha Ghaboush Eldawi Duha Ghaboush Eldawi true true 2018-08-02 The objective of this project is to increase the yield and the quality of in vitro refolded proteins over-expressed in E. coli as insoluble inclusion bodies by optimising the refolding conditions. Therefore, protein refolding additives and regimes like (Glycerol, Gnd-HCl, Dilution, Temperature, and Triton X-100) were investigated to determine their effect on tackling the problems that initiate aggregation in refolding media. The study identified Glycerol as an unsuitable aid in the refolding of Con A due to the binding effect it possessed towards the affinity dextran. Moreover, recommended the use of 0.5 M Gnd-HCl concentration as the initial point that guarantee suitable refolding yields. The rapid and continuous dilution protocols were proved to be more suitable than the other exploited refolding techniques in improving the refolding yields of Con A. However, rapid dilution was the preferable method to use since it is simple and reproducible. The 30X dilution factor was considered the optimum to enhance the refolding yields. A gradual warm up step with 2 hours ice incubation period instead of 1 hour combined with the above mentioned technique ensured the recovery of most of the refolded Con A. Triton X-100 was unsuitable to aid the refolding of Con A due to its inhibitory effects on essential hydrophobic associations. However, its use as a tool was considered a breakthrough for estimating the time-course of refolding of the lectin. Following the resultant optimised strategy the renaturation yield was improved in our laboratory from a mere 12 to 33 mg/lculture i.e. a 2.75-fold increase, providing that more detailed experiments are conducted to improve the use of Triton X-100 as a tool method using purified proteins with accurately known concentration values. E-Thesis Biophysics. 31 12 2008 2008-12-31 COLLEGE NANME Biosciences COLLEGE CODE Swansea University Master of Philosophy M.Phil 2018-08-16T14:39:02.9105634 2018-08-02T16:24:29.3845914 Faculty of Science and Engineering School of Biosciences, Geography and Physics - Biosciences Duha Ghaboush Eldawi NULL 1 0042474-02082018162457.pdf 10798182.pdf 2018-08-02T16:24:57.3400000 Output 7475795 application/pdf E-Thesis true 2018-08-02T16:24:57.3400000 false
title Optimising the refolding conditions of a recombinant protein.
spellingShingle Optimising the refolding conditions of a recombinant protein.
Duha Ghaboush Eldawi
title_short Optimising the refolding conditions of a recombinant protein.
title_full Optimising the refolding conditions of a recombinant protein.
title_fullStr Optimising the refolding conditions of a recombinant protein.
title_full_unstemmed Optimising the refolding conditions of a recombinant protein.
title_sort Optimising the refolding conditions of a recombinant protein.
author_id_str_mv e8b3692eae92308877907a67dc883673
author_id_fullname_str_mv e8b3692eae92308877907a67dc883673_***_Duha Ghaboush Eldawi
author Duha Ghaboush Eldawi
author2 Duha Ghaboush Eldawi
format E-Thesis
publishDate 2008
institution Swansea University
college_str Faculty of Science and Engineering
hierarchytype
hierarchy_top_id facultyofscienceandengineering
hierarchy_top_title Faculty of Science and Engineering
hierarchy_parent_id facultyofscienceandengineering
hierarchy_parent_title Faculty of Science and Engineering
department_str School of Biosciences, Geography and Physics - Biosciences{{{_:::_}}}Faculty of Science and Engineering{{{_:::_}}}School of Biosciences, Geography and Physics - Biosciences
document_store_str 1
active_str 0
description The objective of this project is to increase the yield and the quality of in vitro refolded proteins over-expressed in E. coli as insoluble inclusion bodies by optimising the refolding conditions. Therefore, protein refolding additives and regimes like (Glycerol, Gnd-HCl, Dilution, Temperature, and Triton X-100) were investigated to determine their effect on tackling the problems that initiate aggregation in refolding media. The study identified Glycerol as an unsuitable aid in the refolding of Con A due to the binding effect it possessed towards the affinity dextran. Moreover, recommended the use of 0.5 M Gnd-HCl concentration as the initial point that guarantee suitable refolding yields. The rapid and continuous dilution protocols were proved to be more suitable than the other exploited refolding techniques in improving the refolding yields of Con A. However, rapid dilution was the preferable method to use since it is simple and reproducible. The 30X dilution factor was considered the optimum to enhance the refolding yields. A gradual warm up step with 2 hours ice incubation period instead of 1 hour combined with the above mentioned technique ensured the recovery of most of the refolded Con A. Triton X-100 was unsuitable to aid the refolding of Con A due to its inhibitory effects on essential hydrophobic associations. However, its use as a tool was considered a breakthrough for estimating the time-course of refolding of the lectin. Following the resultant optimised strategy the renaturation yield was improved in our laboratory from a mere 12 to 33 mg/lculture i.e. a 2.75-fold increase, providing that more detailed experiments are conducted to improve the use of Triton X-100 as a tool method using purified proteins with accurately known concentration values.
published_date 2008-12-31T03:53:02Z
_version_ 1763752628703985664
score 11.028886

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