Site-directed Mutagenesis Of Humanization Of Selenium-Containing Single-Chain Abzyme

Reactive oxygen species (ROS) are products of the normal metabolic activities ofaerobic cells and are thought to be the cause of many diseases. ROS include freeradicals such as superoxide anion (O2-·) and hydroxyl radical (·OH), as well as nonradicalintermediates such as hydrogen peroxide (H2O2), hydroperoxide (ROOH), nitric oxide(NO) and singlet oxygen (1O2). Under normal condition, there is a balance between theproduction of ROS and their scavenging. In certain pathogenic states the production ofROS is enhanced and excess ROS damage all cellular components, such as DNA, RNA,proteins and lipids, eventually resulting in ROS –mediated diseases. Examples of suchdiseases are reperfusion injury, inflammation, age-related diseases, neuronal apoptosis,and cancer. To protect themselves from oxidative infury, aerobic cells have anenzymatic and nonenzymatic defense system. The enzymatic antioxidant system ismainly composed of glutathione peroxidase (GPX), catalase (CAT), and superoxidedismutase (SOD). The nonenzymatic antioxidant system includes vitamin E, ascorbate,glutathione (GSH) and uric acid.GPX is a well-know selenoenzyme that functions as an antioxidant and catalyzes thereduction of harmful peroxide by glutathione and protects cells against oxidative damage.Antioxidants are very useful for biological bodies, and have potential for curingROS-related diseases. However, due to the shortcomings of native GPX (low activity,low solubility in water, short half-lives and proteolytic digestion), mimics with high GPXefficiency were prepared by many biochemists. Our laboratory has successfullygenerated several mouse catalytic antibodies, and expressed mouse single-chain variableregion antibodies in E.coli by protein engineering and found high GPX efficiencies bychemical modified serine into selenocysteine (Sec). Although some antibodies frommurine sources have good affinity and showed excellent results in animal models, theproblem with human antimurine (HAMA) antibodies must be resolved.Our laboratory reformed the mouse single-chain varable region antibody (scfv)produced by protein engineering to construct a chimeric library that contained bothconservative fragments of mouse scfv and incorporated the human homologous fragments.Though the selection of phage display library, we acquired the positive phage clone B14that had higher signal of ELISA. The gene fragments of B14 were amplified by PCRand cloned into the expressing vector pPelB. Then pPelB-scfv were transformed intoE.coli Rosetta to express, and was purified and indentified, Sec was incorporated into theprotein by chemical mutation. The GPX activity of the scfv was 880 U/μmol which waslower than expected. The sequence anlysis showed that there were three nucleotidesdifferent from the sites designed,apart located in the Gln27, Val190 of VL and Lys13 ofVH. Thought that the low activity of GPX was dued to the change of DNA sequenceswhich made the conformation of scfv to change .In this study, our strategy was to modify the dismatching bases by overlap PCR whichis a method of sited-directed mutagenesis. The sequence analysis showed that thedismatching bases have been modified. We amplified the correct sequence of scfv byPCR, and cloned to the subcloning vector pGEM-T Easy, seleted and identified thepositive clones. Then amplified the target gene by PCR, cloned to the expressing vetorpPelB vector, identified the positive cloned by restriction mapping and sequence analysis,transforamted into E.coli to decide the optimal strain and expressing conditions byorthogonal experiments. The expressing protein were puried by CM-sepharose Fast Flowand Ni2+ metal ion affinity chromatography, and identified by Western-blot. Sec wasincorporated into the protein by chemical mutation. The GPX activity of the single-chainantibody was 1680 U/μmol, which is 2-fold that of unmutation. The scientific research onthe antibody humanization in this decade suggested that the foundation of antibodyhumanization is the acquirement of the primary structure and the charification of theregularity of the residues'interaction. This study plays a important role in researching thehumanization of selenium-containing single-chain antibody in future, and provide sometheoretical and practical basis for clinical treatment...