Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626332
Title: Protein PEGylation on protein folding
Author: Ginn, C. L.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Abstract:
E.coli is one of the most widely utilised hosts for protein expression due to its rapid growth, low production costs and high product yields. Often proteins are deposited as insoluble inclusion bodies that later require refolding to achieve biological activity. As a result of misfolding and aggregation for many proteins refolding is the yield limiting step in their production. Relevant therapeutic proteins obtained from E.coli include the α-helical barrel proteins (e.g. interferon-α2). Many proteins derived from E.coli are further modified after refolding by the covalent conjugation of poly(ethylene glycol) (PEG). This is known as PEGylation and several PEGylated α-helical barrel proteins are now routinely used in the clinic. PEGylation is used to address the short circulation half-life, immunogenicity and poor stability associated with many protein-based therapeutics. Our method of PEGylation is site specific. Conjugation occurs by bis-alkylation and takes advantage of the presence of the two free thiols from native disulfide bonds that have been reduced. The conjugated product has PEG linked to the protein through a three-carbon bridge spanning the two thiols derived from the native disulfide. Currently proteins are first purified and then a PEG reagent is used to covalently conjugate PEG to the protein. The PEG-protein conjugate is then purified. This means the protein has to be purified twice which can reduce yields. PEGylating the protein during its initial refolding would avoid the need of two downstream purification processes resulting in a more efficient process with an improved product yield. Therefore the aim of this project is to integrate the process of protein folding and PEGylation to make the production of PEGylated proteins more economically viable allowing their widespread use in the clinic. In this project the following hypotheses will be tested i) Reducing the number of purification steps that need to be performed to improve the overall yield of recovered protein, ii) The ability of PEG to impart the properties of a glycosyl group or a chaperone and protect the protein against aggregation during the folding process, iii) our method of PEGylation in particular should promote the formation of the protein’s disulfides bond and therefore the protein’s thermodynamically stable native state and iv) that the specificity of the conjugation can still be maintained despite the exposure of more sites of conjugation. Here we examine this process with different model alpha helical barrel proteins namely leptin, IFN-β and EPO. In each case the protein was denatured and fully reduced then refolded in the presence of a thiol specific, bis-alkylation PEG reagent allowing us to effectively capture the cysteine thiols during the refolding process. For IFN-β which is highly prone to aggregation, refolding yields in the presence of the PEG reagent were much improved suggesting that our method of PEGylation had a stabilising effect on the protein structure during refolding. This improved stability was also found to benefit the protein after PEGylation. Isolation of the purified PEGylated IFN- conjugate could be achieved in a single purification step in good yield. A similar activity to that generated on PEGylation of the fully folded protein was observed suggesting that for a protein with an accessible disulfide PEGylation did not significantly affect its folding. Some work was also carried out on RNase A and T1 which contain multiple inaccessible disulfides. In this instance PEGylation appeared to hinder the refolding process either by sterically hindering the formation of the protein’s native structure or by incorrect disulfide bond formation. The work described herein therefore suggests that it is possible to refold and PEGylate proteins within a single step but the effectiveness of this approach is likely to be protein dependant.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.626332  DOI: Not available
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