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Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria—Application to a single-chain Fv fragment

Paper ID Volume ID Publish Year Pages File Format Full-Text
21221 43212 2010 6 PDF Available
Title
Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria—Application to a single-chain Fv fragment
Abstract

The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichiacoli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions.

Keywords
EDTA, ethylenediaminetetraacetic acid; EGFR, epidermal growth factor receptor; Fab, antigen binding fragment; Fv, variable fragment; mPEG, maleimide-PEG; scFv, single-chain Fv; Mw, molecular weight; PEG, polyethylene glycol; SDS–PAGE, sodium dodecyl sulfa
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Integration of PEGylation and refolding for renaturation of recombinant proteins from insoluble aggregates produced in bacteria—Application to a single-chain Fv fragment
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 109, Issue 5, May 2010, Pages 447–452
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