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Updated: Oct 24, 2026
Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Published on: July 5, 2018
Production, crystallization and diffraction to atomic resolution of an antibody Fv specific for the blood-group A
S I Patenaude1, C R MacKenzie, D Bilous
1Department of Biochemistry, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.
The histoblood-group ABO carbohydrate antigens are well known as important factors in blood transfusions, but they can also act as receptors for infectious agents and have been implicated in susceptibility to certain carcinomas. A single-chain variable-domain antigen-binding fragment (scFv) gene based on the known sequence of an anti-blood-group-A monoclonal antibody (AC1001) has been synthesized and expressed in Escherichia coli. The purified scFv preparation existed primarily in the monomeric form but also contained large amounts of dimeric and higher oligomeric forms. The corresponding variable-domain antigen-binding fragment (Fv) was generated by cleaving the VL-VH linker with subtilisin, and its activity was demonstrated by surface plasmon resonance with an immobilized bovine serum albumin-A-trisaccharide conjugate (KD = 290 microM). AC1001 Fv crystals grown in the presence of N-acetylgalactosamine diffracted to 0.93 A resolution. This is the first reported example of a crystal of an antibody antigen-binding fragment diffracting to atomic resolution.
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