The Construction Of Multifunctional Antioxidant Enzyme Mimic

Reactive oxygen species (ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage various biomacromolecules including RNA, DNA, protein, sugars and lipids, and therefore results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, age-related diseases, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. Due to their pivotal role in scavenging ROS, the enzymatic antioxidant system could act as promising antioxidant drug. However, GPX has some drawbacks such as solution instability, limited cellular accessibility, immunogenicity, short half-lives, costs of production, and proteolytic digestion. And elevation of some kinds of SOD could cause disorder in organisms when other antioxidant enzymes such as GPX and CAT keep constant. Those factors limit the pharmacological use of the naturally occurring enzymes. Scientists have made a great deal of efforts to study their catalytic mechanisms as well as their relationships between them, and prepared many mimics that have the antioxidant ability. But each one of these mimixcs imitated a single enzyme. Actually, the antioxidant enzymes must cooperate with each other to effectively scavenge reactive oxygen species (ROS) because anyone of them can not scavenge all forms of ROS alone. For example, SOD can scavenge O2-·, but the product, H2O2, may still distroy the biomolecules. And GPx can reduced H2O2 to H2O by oxiditing GSH to GSSG, but it can do nothing to O2-·. In addition, the destroyed biomolecules by ROS are still the berden of cells, and it is very important to repair or scavenge them for cells. GST can repair or scavenge the destroyed biomolecules and regenerate S-thiolated protein destroyed by oxidants. In organism, there is a mini system, which is composed from SOD, GPx, GST and glutathione reductase (GR), and plays roles in antioxidation, detoxification and repair of injury. SOD catalyzes dismutation of O2-? to H2O2, GPx reduces H2O2 to H2O and GST repairs the macromolecules oxidized by ROS and regenerate S-thiolated protein.The available information from structural biology indicates that most proteins arise by limited modifications of preexisting protein scaffolds acquiring novel functional properties by recombination of preexisting modules such as amino acid substitution, insertion or deletion of peptide segments, or fusion of different structural domains. This principle can be exploited in the redesign of existing enzymes for novel efficiently antioxidant functions. Based on this principle and the mimics prepared recently of SOD and GPx, we designed a bifunctional enzyme with activity of GPx and SOD by the methods of genetic engineering. And we further construct a SOD, Gpx and GST trifunctional enzyme model by genetic fusion technology.1.Design of a bifunctional enzyme mimic of SOD and GPxAs the main antioxidant enzymes, SOD and GPx can not only synergistic reaction, but also make up of a group to protect each other. In order to imitate the synergism of the enzymes, we designed a bifunctional enzyme with activity of SOD and GPx. Herein, on the basis of the previous 17-mer peptide mimics of SOD and 15-mer peptide mimic of GPx, we designed a new polypeptide, in which the two polypeptides were redesigned by means of structure predication software and linked by a 3-mer peptide linker to eliminate the spatial hindrance between them.2.Design of a trifunctional enzyme with activity of SOD, GPx and GSTa DNA sequence encoding the bifunctional enzyme mimic was deduced and synthesized based on the bias codons of E. coli and cloned into pGEX-2T plasmid vector just at the downstream side of the GST gene. The recombinant pGEX-2T plasmid vector was transformed into E. coli strain BL21 (DE3) to express the goal protein, then the GST fusion protein was purified by affinity chromatography on glutathione Sepharose 4B. The expressed fusion protein exhibited catalytic activities of GST, SOD and GPx after incorporation of copper ions and Selenocysteins. SOD catalyzes O2?- to H2O2, GPx reduces H2O2 to H2O and GST repairs the macromolecules oxidized by ROS and regenerate S-thiolated protein.To our knowledge, this is the first trifunctional antioxidant enzyme mimic prepared thoroughly by genetic engineering so far. Other than chemically artificial enzymes, the trifunctional enzyme was generated by genetic fusion technology, and laid a steady basis both for detailed studies on the cooperation mechanism of these enzymes and for its pharmacological development as an antioxidant.3.Design of the bifunctional enzyme mimic polymers in seriesMost of the native antioxidants are oligomer, but all of the antioxidant enzyme mimics prepared in days gone by have a monomer structure. And this may be the reason of why these mimics have a much lower activity than native enzymes. Herein, we designed a series of bifunctional enzyme mimic polymers in series by linking two or several bifunctional enzyme mimics. In order to eliminate the spatial hindrance between these bifunctional enzyme mimics. one or two thrombin site were inserted between them. Thus the polymers can imitate the oligomer structure of the native enzymes. It is worth to note that these polyers can be cut into monomer bifunctional enzyme mimics by thrombin. We constructed the gene encoden the bifunctional enzyme mimic polymers in series by the technologies of PCR and enzymic linkage cloned it into plasmid pPelB, then the recombinant pPelB plasmid vector was transformed into E. coli strain BL21 (DE3) to express the goal protein induced by IPTG. The goal protein can be purified by Ni-chelate chromatography easily because the pPelB plasmid contains a His tag.