In general, proteins with structural disulfides cannot be expressed in the reducing environment of the cellular cytoplasm. To overcome this folding problem, we have previously engineered stabilizing mutations, predicted from a consensus sequence analysis, into isolated immunoglobulin VL domains. Here we show that such domains can be used as a framework in the construction of a functional heterodimeric Fv fragment, which was expressed solubly, with high yield in the cytoplasm of Escherichia coli. This designed catalytic intrabody, obtained from grafting the combining site of the esterolytic antibody 17E8, is active in the oxidized and the reduced state. Its construction required no special features on the part of the immunoglobulin, no single-chain linker and introduced no non-natural sequence motifs. The potential to design intrabodies with the recognition sequences of arbitrary immunoglobulins opens novel opportunities for gene therapy, cell biology, metabolic engineering and antibody biotechnology.

The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of beta-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased beta-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design.