Intrabody construction and expression. I. The critical role of VL domain stability.
We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations. These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue. Transcription from the strong T7 promoter 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 aggregation. 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.
Ohage
E
E
Genzentrum der Ludwig-Maximilians-Universität, München, Germany.
Steipe
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D004220:Disulfides; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; D008715:Methionine; D003545:Cysteine