title: Intrabody construction and expression III: engineering hyperstable V(H) domains.
abstract: The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin V(L) domains. Here we apply rational stability engineering by consensus sequence analysis to V(H) domains. Isolated V(H) domains tend to aggregate more easily than V(L) domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the V(H) domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20-fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated V(H) domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the V(L) domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.
authors.affiliation: Genzentrum der Ludwig-Maximilians-Universität, München, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Wirtz
authors.forename: B
authors.initials: B
authors.lastname: Steipe
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004355:Drug Stability; D004926:Escherichia coli; D006358:Hot Temperature; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins