Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbB-2 receptor.
We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
Wels
W
W
Friedrich Miescher Institute, Basel, Switzerland.
Harwerth
I M
IM
Zwickl
M
M
Hardman
N
N
Groner
B
B
Hynes
N E
NE
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005798:Genes, Bacterial; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases
D000469:Alkaline Phosphatase; D000911:Antibodies, Monoclonal; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases