Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604856
Title: Purification and refolding strategies for intensifying inclusion body processing
Author: Hutchinson, M. H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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Abstract:
Adsorptive protein refolding was investigated as an alternative to batch-dilution refolding for two model poly-histidine tagged proteins: Δ5-3-ketosteroid isomerase (KSI-(His6)) and glutathione S-transferase (GST-(His6)). Refolding was performed by removing the denaturant while the protein was adsorbed to metal affinity adsorbents through the C-terminal tag. For GST-(His6), the concentration at which the protein was refolded could be increased 4-fold compared to dilution refolding without reducing the yield of correctly refolded, enzymatically active protein. The overall refolding yield for KSI-(His6) adsorbed at 3 mg/mL adsorbent increased from50% (for a single step process) to 75% with three additional iterations of the refolding operation. An adsorption refolding step was then incorporated into an intensified process for obtaining purified and correctly refolded KSI-(His6) from E.coli inclusion bodies. Chemical extraction was used to disrupt the cells and simultaneously solubilize the inclusion bodies.  An expanded bed adsorption (EBA) column utilizing immobilized metal affinity interactions was used to purify denatured KSI-(His6) directly from the chemical extract. This integrated process achieved a very high purity (99%) and recovery (89%) of KSI-(His6), with efficient utilization of the adsorbent matrix (9.7 mg KSI-(His6)/mL adsorbent). The denaturant was then removed to refold the protein while it remained adsorbed in the column. 10% of the KSI-(His6) expressed in the inclusion body was recovered as biologically active product using this highly simplified process. The process yield was further increased to 19% by performing a second iteration of the refolding step. This efficient, generic, and scale invariant process has the potential to improve the economic advantage of using the highly productive E. coli host organism for recombinant protein expression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.604856  DOI: Not available
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