The aggregation of cytochrome C may be linked to its flexibility during refolding

James Austerberry, Daniel Belton

Research output: Contribution to journalArticle

Abstract

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules.
Original languageEnglish
Number of pages4
Journal3 Biotech
Volume6
Issue number33
Early online date14 Jan 2016
DOIs
Publication statusPublished - Jun 2016

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protein aggregates
Cytochromes
cytochromes
cytochrome
Period Circadian Proteins
Fluorescence Polarization
protein
Protein C
Urea
Proteins
proteins
biopharmaceuticals
protein folding
urea
folding
fluorescence
dilution
anisotropy
Protein Aggregates

Cite this

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The aggregation of cytochrome C may be linked to its flexibility during refolding. / Austerberry, James ; Belton, Daniel.

In: 3 Biotech, Vol. 6, No. 33, 06.2016.

Research output: Contribution to journalArticle

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T1 - The aggregation of cytochrome C may be linked to its flexibility during refolding

AU - Austerberry, James

AU - Belton, Daniel

PY - 2016/6

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AB - Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules.

KW - protein refolding

KW - molten globule

KW - fluorescence anisotropy

KW - protein aggregation

KW - protein flexibility

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