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The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli


Wall, JGerard; Plückthun, Andreas (1999). The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli. Protein Engineering, Design & Selection, 12(7):605-611.

Abstract

In a systematic study of the periplasmic folding of antibody fragments in Escherichia coli, we have analysed the expression of an aggregation-prone and previously non-functional anti-phosphorylcholine antibody, T15, as a model system and converted it to a functional molecule. Introduction of heavy chain framework mutations previously found to improve the folding of a related antibody led to improved folding of T15 fragments and improved physiology of the host E.coli cells. Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved folding and bacterial host physiology, but no improvement was seen in the wild type, suggesting the existence of a hierarchy in sequence positions leading to aggregation. Rational mutagenesis of the T15 light chain led to the production of functional T15 fragments for the first time, with increased levels of functional protein produced from VH manipulated constructs. We propose that a hierarchical analysis of the primary amino acid sequence, as we have described, provides guidelines on how correctly folding, functional antibodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation

Abstract

In a systematic study of the periplasmic folding of antibody fragments in Escherichia coli, we have analysed the expression of an aggregation-prone and previously non-functional anti-phosphorylcholine antibody, T15, as a model system and converted it to a functional molecule. Introduction of heavy chain framework mutations previously found to improve the folding of a related antibody led to improved folding of T15 fragments and improved physiology of the host E.coli cells. Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved folding and bacterial host physiology, but no improvement was seen in the wild type, suggesting the existence of a hierarchy in sequence positions leading to aggregation. Rational mutagenesis of the T15 light chain led to the production of functional T15 fragments for the first time, with increased levels of functional protein produced from VH manipulated constructs. We propose that a hierarchical analysis of the primary amino acid sequence, as we have described, provides guidelines on how correctly folding, functional antibodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation

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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
Language:English
Date:1 July 1999
Deposited On:20 Sep 2018 14:19
Last Modified:04 Oct 2018 11:18
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/12.7.605
Related URLs:https://www.swissbib.ch/Search/Results?lookfor=nationallicenceoxford101093protein127605 (Library Catalogue)

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