A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.
A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.
Tai
M S
MS
Creative BioMolecules, Inc., Hopkinton, Massachusetts 01748.
Mudgett-Hunter
M
M
Levinson
D
D
Wu
G M
GM
Haber
E
E
Oppermann
H
H
Huston
J S
JS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004077:Digoxin / Q000276:immunology* / Q000378:metabolism; D005813:Genes, Synthetic; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004077:Digoxin