Biotechnological production of a stereoselective scFv antibody fragment

Includes bibliographical references.Antibodies are proteins of the immune system that exhibit exquisite specificity to their binding partners, called antigens. Binding occurs at the hypervariable region (Fv), which is capable of discriminating even enantiomeric molecules. By targeting on the DNA sequences that code for Fv and expressing it as a protein, the binding properties of an antibody can be assessed. In this project, the heavy and light chains of the Fv region (Vh and V[_) of an anti-amino acid antibody (anti-phenylalanine 67.36) were linked using a short nucleotide linker and four primers designed from previously determined sequences of Vh and V|_. The resulting V|-H¡nker-V|_(HIL) construct - a single chain variable fragment (67.36 HIL scFv) - was inserted into a TOPO cloning vector and transformed into E. coli BL 21 expression cells. 1 L cultures were grown at 37°, 30° and 27 °C. Cells were collected, resuspended and lysed to release the protein of interest. Isolation and purification was performed using a metal (cobalt) affinity chromatography resin. Characterization by SDS-PAGE indicated a correlation between incubation temperature and extent of expression. Noncompetitive ELISA tests revealed a functional 67.36 HIL scFv. However, further tests e.g., competitive ELISA, must be carried out to confirm an enantiomeric preference.B.S. (Bachelor of Science