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

In a systematic study of the periplasmic folding of antibody fragments in E scherichia coli, we have analysed the expression of an aggregation-prone an d previously non-functional anti-phosphorylcholine antibody, T15, as a mode l system and converted it to a functional molecule. Introduction of heavy c hain framework mutations previously found to improve the folding of a relat ed antibody led to improved folding of T15 fragments and improved physiolog y of the host E.coli cells. Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved foldi ng and bacterial host physiology, but no improvement was seen in the wild t ype, suggesting the existence of a hierarchy in sequence positions leading to aggregation. Rational mutagenesis of the T15 light chain led to the prod uction of functional T15 fragments for the first time, with increased level s of functional protein produced from V-H manipulated constructs. We propos e that a hierarchical analysis of the primary amino acid sequence, as we ha ve described, provides guidelines on how correctly folding, functional anti bodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation.