| 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 recognize and bind substrate, and that the catalytic group is at a correct position to interact 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 antibodies1 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 an scFv of monoclonal antibody 2F3 (scFv2F3) in E. coli, which has a GSH affinity. These antibodies may show (or show upon modification) some enzymic activities with glutathione as substrate.The GST Properties of Soluble ScFv2F3ScFv2F3 was expressed in inclusion body form. It needs to be denatured and renatured to get the functional protein. We cultured a new expression strain that can express soluble scFv2F3.The soluble proteins were purified by Ni2+-IMAC affinity chromatographyand by HPLC further. The finally obtained protein was identified with western blot. We implemented the expression of single-chain antibody as a soluble protein.Glutathione S-transferases (GSTs) are one kind of enzymes, one of which substrates is glutathione. They are a superfamily of multifunctional enzymes involved in the cellular detoxification. They achieve this by conjugating the tri-peptide glutathione (GSH) to a wide range of endobiotic and xenobiotic electro-philic compounds. Although they all have similar structure and topology of active center, they have very different catalytic residues including Tyr, Ser and Cys. In the scFv2F3 molecule, there are many Tyr, Ser and Cys residues. Especially, the Tyr, Ser and Cys residues in CDRs may approach to the -SH of glutathione and catalyze the conjugation of glutathione with endobiotic and xenobiotic electro-philic compounds.We assayed the GST activities of scFv2F3 to several electrophilic compounds and found that scFv2F3 exhibited GST activity to them. The GST activities to l-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ETHA), 1-bromo-4-nitrobenzene and cumene hydroperoxide are 0.997 U/mg, 0.109 U/mg, 3.646 U/mg and 0.928 U/mg respectively. Because 1-bromo-4-nitrobenzene is the marker substrate of 0-class GST, the result suggested that the catalytic residue in scFv2F3 is Ser. Modified the Ser residues in scFv2F3, scFv2F3 lost GST activity, which improved that the catalytic residue in scFv2F3 is Ser.We have researched the GST properties of scFv2F3 with CDNB, a relatively nonspecific GST reference substrate. The optimal pH and temperature for scFv2F3 catalyzed conjugation of GSH with CDNB was found to be pH 7.9 and 44C respectively. The steady-state kinetic studies showed that the Km of reduced glutathione and the Km of l-chloro-2,4-dinitrobenzene are closed to those of native glutathione S-transferases, indicating that scFv2F3 had a strong affinity for the substrates. But this result caused a problem that the velocity of catalytic conversion was delayed and the kcat was low. So to acquire a catalytic antibody, the design of hapten is important and it is not fundamental to choose antibody with the strongest affinity to hapten.These data suggested that- it is possible to alter the structure o...
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