A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti-E-selectin monoclonal antibody to activated human endothelial cells.
Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5' to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the VH-Linker-VL 32-33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.
Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
R
R
Casalvilla
M
M
Dueñas
M
M
Ayala
S
S
Cruz
L
L
Cruz
W A
WA
Buurman
J V
JV
Gavilondo
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D019040:E-Selectin / Q000276:immunology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D019040:E-Selectin; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha