Production and secretion of a bifunctional staphylococcal protein A::antiphytochrome single-chain Fv fusion protein in Escherichia coli.
A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.
Gandecha
A R
AR
Department of Botany, University of Leicester, UK.
Owen
M R
MR
Cockburn
B
B
Whitelam
G C
GC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010834:Phytochrome / Q000276:immunology*; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D010834:Phytochrome; D004247:DNA