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Dilution Versus Dialysis: A Quantitative Study of the Oxidative Refolding of Recombinant Human Alpha-fetoprotein

Paper ID Volume ID Publish Year Pages File Format Full-Text
19613 43084 2006 9 PDF Available
Title
Dilution Versus Dialysis: A Quantitative Study of the Oxidative Refolding of Recombinant Human Alpha-fetoprotein
Abstract

Alpha-fetoprotein (AFP) is a commercially important polypeptide with important diagnostic, physiological and immunomodulatory functions. Previous studies into the refolding of this macromolecule are contradictory, and variously suggest that AFP denaturation may be irreversible or that refolding may be achieved by reducing denaturant concentration through dilution but not dialysis. Importantly, these same previous studies do not provide quantitative metrics by which the success of refolding, and the potential for bioprocess development, can be assessed. Moreover, these same studies do not optimize and control refolding redox potential — an important factor considering that AFP contains 32 cysteines which form 16 disulfide bonds. In this current study, a quantitative comparison of recombinant human AFP (rhAFP) refolding by dilution and dialysis is conducted under optimized redox conditions. rhAFP refolding yields were .35% (dialysis refolding) and >75% (dilution refolding) as assessed by RP-HPLC and ELISA, with structural similarity to the native state confirmed by UV spectroscopy. Dialysis refolding yield was believed to be lower because the gradual reduction in denaturant concentration allowed extended conformational searching, enabling more time for undesirable interaction with other protein molecules and/or the dialysis membrane, leading to a sub-optimal process outcome. Significant yield sensitivity to redox environment was also observed, emphasizing the importance of physicochemical optimization. This study demonstrates that very high refolding yields can be obtained, for a physiologically relevant protein, with optimized dilution refolding. The study also highlights the quantitative metrics and macromolecular physical spectroscopic ‘fingerprints’ required to facilitate transition from laboratory to process scale.

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Dilution Versus Dialysis: A Quantitative Study of the Oxidative Refolding of Recombinant Human Alpha-fetoprotein
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Publisher
Database: Elsevier - ScienceDirect
Journal: Food and Bioproducts Processing - Volume 84, Issue 1, March 2006, Pages 9-17
Authors
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us