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 i...

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Bibliographic Details
Published in:Biochemistry (Easton) 1990-09, Vol.29 (35), p.8024-8030
Main Authors: Tai, Mei Sheng, Mudgett-Hunter, Meredith, Levinson, Douglas, Wu, Gay May, Haber, Edgar, Oppermann, Hermann, Huston, James S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Antibodies, Monoclonal - genetics
Antibodies, Monoclonal - metabolism
Antibody Affinity
Base Sequence
Binding Sites, Antibody
Biological and medical sciences
Digoxin - immunology
Digoxin - metabolism
Fundamental and applied biological sciences. Psychology
Gene expression
Genes, Synthetic
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - metabolism
Immunoglobulin Fc Fragments - metabolism
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Peptide Fragments - metabolism
Protein Binding
Protein Conformation
Protein Engineering
Recombinant Fusion Proteins - metabolism
Staphylococcal Protein A - genetics
Staphylococcal Protein A - metabolism
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00487a005