Intrabody construction and expression. II. A synthetic catalytic Fv fragment.
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.
Ohage
E C
EC
Genzentrum der Ludwig-Maximilians-Universität, München, Germany.
Wirtz
P
P
Barnikow
J
J
Steipe
B
B
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain; D016384:Consensus Sequence / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D017151:Antibodies, Catalytic; D004220:Disulfides; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv