title: The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli.
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 V(H) 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.
authors.affiliation: Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190,CH-8057 Zürich, Switzerland.
authors.forename: J G
authors.initials: JG
authors.lastname: Wall
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010767:Phosphorylcholine / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
chemical_list: D000907:Antibodies, Bacterial; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010767:Phosphorylcholine