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Folding and aggregation of a multi-domain engineered immunotoxin ☆

Paper ID Volume ID Publish Year Pages File Format Full-Text
3213 159 2013 7 PDF Available
Title
Folding and aggregation of a multi-domain engineered immunotoxin ☆
Abstract

•Two-domain fusion protein between an antibody and toxin domain.•Antibody domain is shown to be the more aggregation-prone region of the protein.•Antibody domain must be refolded first, at intermediate denaturant concentrations, in order to suppress aggregation during refolding.•A step-wise refolding process, guided by the experimental three-state unfolding thermodynamics of the system, is successful in greatly suppressing aggregation during technical refolding.

Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (VH/VL) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and VH/VL domains had considerably different unfolding free energies (ΔGUNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the VH/VL folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the VH/VL domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the VH/VL domain played a key role in aggregation of this antibody fusion protein.

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Keywords
Aggregation; Biophysical chemistry; Protein denaturation; Protein recovery
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Folding and aggregation of a multi-domain engineered immunotoxin ☆
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 81, 15 December 2013, Pages 8–14
Authors
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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