Preparation, characterization and crystallization of recombinant fragment of anti-CD3 antibody MEM-57
Jana Písačková, Vlastimil Král, Magdaléna Hořejší, Milan Fábry and Pavlína Řezáčová
Institute of Molecular Genetics of the ASCR,
v.v.i., Flemingovo nam. 2, Prague 6,
166 37, Czech Republic
Monoclonal antibody MEM-57 recognizes CD3
surface glycoprotein, which associates with T-cell receptor and mediates
activation signal transduction. CD3 antigen is expressed on peripheral blood T-lymphocytes
. Antibody MEM-57 can, therefore, be used as a part of the „Bispecific T-cell
Single‑chain variable fragment of MEM‑57 (scFv MEM-57) was produced in E. coli BL21(DE3) from a pET22b vector; the product, targeted into the periplasmic space by the pelB leader sequence, contained c‑myc and His5 tag at the C-terminus. The purification consisted in two steps, nickel chelation affinity chromatography and ion‑exchange chromatography. The antigen binding activity was confirmed by flow cytometry.
Crystallographic studies were initiated, as
the structural information on scFv MEM‑57 would be useful for
humanization of the antibody. This poster focuses mainly on experiments aiming at
pre-crystallization analysis and the improvement of protein crystallizability.
Size‑exclusion chromatography, dynamic light scattering and differential
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