FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W204948315> ?p ?o ?g. }

• W204948315 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." @default.
• W204948315 created "2016-06-24" @default.
• W204948315 creator A5054110389 @default.
• W204948315 date "2013-09-28" @default.
• W204948315 modified "2023-09-23" @default.
• W204948315 title "Protein PEGylation on Protein Folding" @default.
• W204948315 hasPublicationYear "2013" @default.
• W204948315 type Work @default.
• W204948315 sameAs 204948315 @default.
• W204948315 citedByCount "0" @default.
• W204948315 crossrefType "dissertation" @default.
• W204948315 hasAuthorship W204948315A5054110389 @default.
• W204948315 hasConcept C10138342 @default.
• W204948315 hasConcept C134306372 @default.
• W204948315 hasConcept C147816474 @default.
• W204948315 hasConcept C162324750 @default.
• W204948315 hasConcept C178790620 @default.
• W204948315 hasConcept C180577832 @default.
• W204948315 hasConcept C181199279 @default.
• W204948315 hasConcept C185592680 @default.
• W204948315 hasConcept C197336794 @default.
• W204948315 hasConcept C204328495 @default.
• W204948315 hasConcept C21951064 @default.
• W204948315 hasConcept C2776964284 @default.
• W204948315 hasConcept C2777516009 @default.
• W204948315 hasConcept C2779149719 @default.
• W204948315 hasConcept C2779201268 @default.
• W204948315 hasConcept C33923547 @default.
• W204948315 hasConcept C54400483 @default.
• W204948315 hasConcept C55493867 @default.
• W204948315 hasConceptScore W204948315C10138342 @default.
• W204948315 hasConceptScore W204948315C134306372 @default.
• W204948315 hasConceptScore W204948315C147816474 @default.
• W204948315 hasConceptScore W204948315C162324750 @default.
• W204948315 hasConceptScore W204948315C178790620 @default.
• W204948315 hasConceptScore W204948315C180577832 @default.
• W204948315 hasConceptScore W204948315C181199279 @default.
• W204948315 hasConceptScore W204948315C185592680 @default.
• W204948315 hasConceptScore W204948315C197336794 @default.
• W204948315 hasConceptScore W204948315C204328495 @default.
• W204948315 hasConceptScore W204948315C21951064 @default.
• W204948315 hasConceptScore W204948315C2776964284 @default.
• W204948315 hasConceptScore W204948315C2777516009 @default.
• W204948315 hasConceptScore W204948315C2779149719 @default.
• W204948315 hasConceptScore W204948315C2779201268 @default.
• W204948315 hasConceptScore W204948315C33923547 @default.
• W204948315 hasConceptScore W204948315C54400483 @default.
• W204948315 hasConceptScore W204948315C55493867 @default.
• W204948315 hasLocation W2049483151 @default.
• W204948315 hasOpenAccess W204948315 @default.
• W204948315 hasPrimaryLocation W2049483151 @default.
• W204948315 hasRelatedWork W1509468073 @default.
• W204948315 hasRelatedWork W1939054711 @default.
• W204948315 hasRelatedWork W1971659890 @default.
• W204948315 hasRelatedWork W1973033448 @default.
• W204948315 hasRelatedWork W1998499505 @default.
• W204948315 hasRelatedWork W1998915545 @default.
• W204948315 hasRelatedWork W2007492537 @default.
• W204948315 hasRelatedWork W2034130222 @default.
• W204948315 hasRelatedWork W2079474827 @default.
• W204948315 hasRelatedWork W2086874318 @default.
• W204948315 hasRelatedWork W2125537720 @default.
• W204948315 hasRelatedWork W2358384169 @default.
• W204948315 hasRelatedWork W2399388661 @default.
• W204948315 hasRelatedWork W2726687315 @default.
• W204948315 hasRelatedWork W2809109008 @default.
• W204948315 hasRelatedWork W2891315072 @default.
• W204948315 hasRelatedWork W3030280621 @default.
• W204948315 hasRelatedWork W2025825854 @default.
• W204948315 hasRelatedWork W2849445775 @default.
• W204948315 hasRelatedWork W2942412248 @default.
• W204948315 isParatext "false" @default.
• W204948315 isRetracted "false" @default.
• W204948315 magId "204948315" @default.
• W204948315 workType "dissertation" @default.