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Facilitated EGFP IBS Refolding By Like-charged Materials

Posted on:2015-05-10Degree:MasterType:Thesis
Country:ChinaCandidate:R ChenFull Text:PDF
GTID:2180330452469850Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Protein refolding is crucial step in the production of genetic engineeringproducts.We have previously found that addition of like-charged media in a refoldingsolution can greatly enhance the refolding of pure proteins by suppressing proteinaggregation. Herein, negatively charged mono-sized microspheres with sulfonicgroups were fabricated to explore the facilitating effect of like-charged media on therefolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies(IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy wasdeveloped to increase the charge density of mono-sized poly(glycidyl methacrylate)(pGMA) beads (2.4μm). Namely, GMA was first grafted onto the beads by graftingpolymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxygroups on the tentacles were converted into sulfonic groups by modification withsodium sulifte. By this fabrication strategy, the charge density of the beads (pGMA-[GMA-SO3-]n3) reached793μmol/g, about2.8times higher than that modifiedwithout prior grafting of the pGMA beads (pGMA-SO3-,285μmol/g).The negatively charged beads of different charge densities were used forfacilitating the refolding of like-charged EGFP from IBs,which were cultivated fromEscherichia coli strain harboring the pET28a-EGFP vector for EGFP. The resultsshow that the refolding yield as well as refolding rate increased with increasing chargedensity, the anti-aggregation effects of urea and like-charged microspheres weresynergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to52.8%removal of positively charged contaminant proteinsin the refolded solution. Finally, reusability of the tentacle beads was demonstrated byrepetitive EGFP refolding and recovery cycles.
Keywords/Search Tags:Mono-sized microsphere, Tentacle besd, Mdoiffication, Negatively charged surface, Protein refolding, Purification
PDF Full Text Request
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