Construct Selenium-containing Single Chain Antibody With GPx Activity Based On Catalytic Module Assembly And Investigate Their Activity

GPx is a well-known selenoenzyme that functions as an antioxidant andcatalyzes the reduction of harmful peroxide by glutathione and protects cells againstoxidative damage. However, native GPx has poor stability and extraction andpurification are troublesome. Therefore, people always focus on research of the GPxmimics. Effective method for producing GPx mimics with high GPx activity is thepreparation of selenium-containing abzyme. A single chain antibody variablefragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and lightchains (VL) of immunoglobulins, connected with a short linker peptide. Compared tothe normal abzyme, scFv is suitable as drugs for its lower molecular weight.In order to imitate the catalytic triad of native glutathione peroxidase(GPx) inantibody GPx mimic, a novel selenium-containing single chain variable fragment(scFv) with glutathione peroxidase activity was constructed via introducing catalytictriad(catalytic module). Based on theoretical evidence, it has been proposed that theside chains scFv-2F3located close to Ser52, were mutated to Trp29and Gln72,respectively to create a catalytic module agrees well with the neighborhood structura1features of native GPx consisting of Trp29(W), Cys52(C), and Gln72(Q). Tovalidate the predictions of the theoretical studies, catalytic module of GPx in antibodymimics for the first time was constructed. The catalytic module composed of Trp29(W), Sec52(U), and Gln72(Q) were synthesized via site-directed mutagenesismethod and expressed in cysteine auxotrophic expression system.Studies of the enzymatic properties have shown that scFv-2F3-29W52U72Qhaven’t GST activity, but the mutant of scFv-2F3-29W52S72Q has shown GSTactivity of43.9U/μmol(CDNB)， a little higher than scFv-2F3(CDNB).Itsnitro-bromobenzene was150.54U/μmol，ETHA was5.02U/μmol.The results indicate that the GPx activity of the scFv-29W52U72Q introduced by the catalyticmodule was significantly higher and was determined to be4235U/μmol, close to thenatural GPx activity, and other mutants’ GPx activity was lower. Further study bymolecular dynamics,it exhibited pH and temperature dependent catalytic activity anda typical ping-pong kinetic mechanism. Therefore, the catalytic module is crucial toenhance the GPx catalytic activity and stability.