For more than 20 years now, periplasmic expression in Escherichia coli has become the standard technology for preparing functional antibody fragments in a rapid and
convenient way (Skerra and Plückthun 1988; Plückthun et al. 1996). The criteria of choosing either the Fab or single-chain Fv fragment (scFv) format, the properties of
suitable expression vectors, as well as the influence of the E. coli strain used have been extensively summarized elsewhere (Plückthun et al. 1996). However, even
when considering all these components and experimental conditions, the yield of recombinant antibody fragments is still highly variable, mainly being a direct
consequence of the primary sequence and its sequence-dependent propensity to lead to aggregation-prone folding intermediates. In general, periplasmic folding is
the yield-limiting step, being strongly influenced by the amino acid composition of the antibody to be expressed (Wörn and Plückthun 2001; Ewert et al. 2004).