Construction of di-scFv through a trivalent alkyne-azide 1,3-dipolar cycloaddition.
Heterofunctional azide and alkyne PEG-linkers have been synthesized and site specifically conjugated to scFv via a reactive thiol functionality; two scFv were coupled by copper catalyzed 1,3-dipolar cycloaddition to make divalent scFv (di-scFv) with an inter-scFv distance defined to provide divalent binding; antigen binding was maintained for the di-scFv construct and increased several times compared to that of the parent scFv; the cycloaddition reaction reported herein represents an important ligation strategy to covalently link macromolecular proteins and retain sensitive structural conformations.
Natarajan
Arutselvan
A
Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California, Davis Medical Center, 1508 Alhambra Boulevard, Sacramento, CA 95816, USA.
Du
Wenjun
W
Xiong
Cheng-Yi
CY
DeNardo
Gerald L
GL
DeNardo
Sally J
SJ
Gervay-Hague
Jacquelyn
J
D000480:Alkynes; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D001386:Azides; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000627:therapeutic use; D007150:Immunohistochemistry; D008297:Male; D015202:Protein Engineering
D000480:Alkynes; D000951:Antigens, Neoplasm; D001386:Azides; D003432:Cross-Linking Reagents; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region