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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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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 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.
Keywords: Biophysics.
College: Faculty of Science and Engineering

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