Single Chain Antibody ScFv2F3 Displays Activity Of Glutathione S-transferase

Enzyme is a wonderful biological macromolecule. It has many special catalytic properties that attract the interests of many scientists. The generation of enzyme-like catalysts continues to be a fundamental goal for biochemists. During working in this research, people found the two principal phenomena that underlie the activity of enzyme: substrate binding and the subsequent intracomplex reaction. When one constructs an enzyme model, it is necessary to consider that enzyme model can both recognize and bind substrate, and that the catalytic group is at a correct position to be helpful for its interacting with the reactive group of substrate in order to promote catalysis. We developed an approach to enzyme redesign that capitalizes on introducing catalytic residues into a substrate-binding site to create an active site capable of catalyzing a chemical reaction. To generate a substrate-binding site, one of the general strategies is standard monoclonal antibody preparation technique. Therefore, the characteristic binding specificity of antibodies offers the potential for unique substrate selectivity by catalytic antibodies (abzyme). Since 1990s, we have generated a series of monoclonal antibodies by means of hydrodoma technique using GSH derivatives as haptens and recently we successfully cloned and expressed single chain fragments of variable region of monoclonal antibody 2F3 (scFv2F3) in E. coli, which showed high affinity for GSH. These antibodies may show (or show upon modification) some enzymic activities with glutathione as substrate. The GST Properties of Soluble ScFv2F3 When scFv2F3 was expressed in inclusion body form,it needs to be denatured and renatured to get the functional protein. We developed a new expression strain that can express soluble scFv2F3. The soluble proteins were purified by Ni2+-IMAC affinity chromatography and by HPLC further. The finally obtained protein was identified with western...