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Engineered turns of a recombinant antibody improve its in vivo folding


Knappik, Achim; Plückthun, Andreas (1995). Engineered turns of a recombinant antibody improve its in vivo folding. Protein Engineering, Design & Selection, 8(1):81-89.

Abstract

Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions

Abstract

Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:National licences > 142-005
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Biotechnology
Physical Sciences > Bioengineering
Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Language:English
Date:1 January 1995
Deposited On:12 Oct 2018 06:52
Last Modified:15 Apr 2021 14:47
Publisher:Oxford University Press
ISSN:1741-0126
OA Status:Green
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1093/protein/8.1.81

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