Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595323
Title: Strategies for expanded bed purification of recombinant protein
Author: Abdullah, N.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Abstract:
True biospecific affinity chromatography, immobilized metal affinity chelating (IMAC) and ion exchange chromatography (IEX) strategies, were investigated for EBA purification of a model system, recombinant glutathione-S-transferase (GST). All three strategies were performed using an unclarified E.coli homogenate containing GST concentration at 20-25 % of the total protein, which represented a large initial degree of purity. True biospecific affinity strategy exploits the naturally occurring affinity between GST and its natural substrate, glutathione (GSH). EBA purification using STREAMLINE GSH adsorbent was very specific and lead to a high purification fold of the target GST. However, it was hampered by a poor dynamic capacity (39 U mL-1) and a low recovery yield (34 %). Although some improvements by using a greater volume of adsorbent in the expanded bed and operating at a lower superficial velocity with concomitant lower degrees of bed expansion were obtained, the overall purification performance remained unsatisfactory. This supports the hypothesis that affinity adsorbents are an unnecessary elaboration where products are already substantially pure. The use of short peptide tags on proteins in conjunction with EBA, using generic adsorbents to facilitate the facile recovery of the target protein, is a promising approach to simplify downstream processing flowsheets. Two IMAC adsorbents, STREAMLINE-IDA and STREAMLINE-NTA loaded with Ni2+ were used to purify GST-(His)6, a construct specifically tagged with a histidine-rich sequence at the C-terminus. Both adsorbents exhibited adsorption performance similar to each other in EBA purification. They remained superior with high dynamic adsorption capacities (IDA at 357 U mL-1 and NTA at 263 U mL-1), high selective separation and highly robust adsorbents, compared to IEX and GSH adsorption systems. However, if the target protein is destined for therapeutic use, it may be necessary to incorporate additional processes to ensure the removal of the histidine-rich tag sequence. Due to the propensity of GST to form multimers, cleavage of the tag from this protein was unsuccessful. Such complications may disfavour the widespread adoption of this approach. However, in other model protein, polyhistidine tagged maltose-binding protein (MBP), successful tag digestion was obtained. The tag removal process was simple and efficient with a high yield of MBP. The simplicity and ability to operate with minimal loss of yield, indicates that the combination of affinity fusions and IMAC strategy for EBA, has great potential for efficient large-scale operation. The use of economic I EX approaches is an attractive alternative to avoid the need for affinity tag fusion consideration. IEX are popular for protein purification due to their robustness and versatility. When using STREAMLINE DEAE, additional purification steps are required to achieve a high degree of product purity, due to their low protein selectivity and some competition for adsorption between GST and  Other Components  in  the feedstock.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.595323  DOI: Not available
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