Intrabody construction and expression. 1. The critical role of V-L domain stability

We have constructed a panel of hyperstable immunoglobulin V-L domains by a rational approach of consensus sequence engineering and combining stabilizi ng point mutations. These prototype domains unfold fully reversibly, even w hen the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunogl obulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excel lent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise V-L stab ility, but this problem can be overcome by changing the amino acid followin g the initiator methionine residue. Transcription from the strong T7 promot er does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained. Elevated temperature shifts protein from a productive folding pathway to a ggregation. The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro. In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains. (C) 1999 Academic Press.