Directed Evolution Of Superoxide Dismutase

Superoxide dismutases play an important part in organisms' defense against highly reactive oxygen species by removing the superoxide anions. According to their metal cofactor, three different classes of superoxide dismutases can be distinguished: Cu,Zn-SOD, Fe-SOD and Mn-SOD. Cu,Zn-SODs are predominantly found in the cytosol and peroxisomes of eukaryotes and have also been detected in chloroplasts and in some bacterial strains. There are two kinds of copper- and zinc-superoxide dismutase: cytosolic Cu,Zn-SOD and extracellular SOD (EC-SOD). EC-SOD and Cu,Zn-SOD catalyze the dismutation action with the same efficiency and have very similar kinetic parameters. Comparison of amino acid sequences has suggested that high homology among Cu,Zn-SODs in different species is parallel to that among cytochrom Cs.As superoxide radicals are involved in the pathogenesis of a variety of diseases, more and more attentions are paid to the clinical application of superoxide dismutases. Cu,Zn-SOD has been shown to be effective for treating inflammation, reducing reperfusion injuries, and decreasing blood pressure in animal model systems.In the experiment, the cDNA deprived from human liver encoding Cu,Zn-SOD is amplified by RT-PCR and sequenced. The 0.49kb DNA fragment obtained was cloned into pBV220 vector to construct a high level expression recombinant plasmid designated pBVSOD. Result from SDS-PAGE shows that a specific protein with a molecular weight of 19KD and 30.17% of the total bacterial proteins appears in E.coli including pBVSOD, which is absent in control bacteria including pBV220.However, the use of human Cu,Zn-SOD in human medicine is greatly limited because of the short circulatory half-life due to kidney clearance. Endothelial cells are enriched with acidic proteoglycans, such as heparan sulfate. Proteins with high affinity for heparin-like proteoglycans have been shown to localize on the outer surface of vascular endothelial cells. The half-life of the HEC-SOD has been prolonged to 10 Hrs. And then, the HEC-SOD gene was isolated from lung tissue using RT-PCR and clonedsuccessfully. But, considerable effort has been devoted, without success, to the expression of HEC-SOD in prokaryotes such as Escherichia coli or yeast. We tested several expression vectors constructed with diverse promoters, such as pBV220 vector with lambda phage PR?L promoter, pET vectors with T? phage promoter and pGEX-2TK vector with Trp-Lac promoters. But all our efforts went to waste.Directed evolution can compress several millennia that nature takes into several months and it is composed of reiterative cycles of error-prone PCR (or StER) followed by screening. According to previous work, three important parts constitute this artificial process, one is construction of mutant libraries, the other is effective screening method on the large scale and the last one is appropriate evolutionary pressure.After five rounds of evolution, we got a lot of mutants containing neutral or negative point mutations on the medium without paraquat. Under artificial oxidative pressures, the results of evolution are similar to those on the natural conditions, but to our excitation, we acquire a useful SOD mutant worthy of further research. DNA sequence proves that it contains four point mutations: Phe45(TTC->TTT), Val87(GTG->GTA), Glul33^Gln(GAA^CAA), and Alal40^Gly(GCT-*GGT). In our opinion, although the first and the second are synonymous mutations, they are in cells' favor because of their higher usage than those of the unchanged in E.coli. In some sense, this phenomenon is natural response to the harsh environment that means high efficiency of translation and more expression. Perhaps it is a good way to increase the intracellular enzyme activity due to our target gene without regulating regions. As far as Alal40?Gly is concerned, we think it may facilitate to form the H-bond for more stable second structure. But for us, the most importance of these four point mutations is the third (Glul33?Gln). Because Glul33 is just on the verge of the pocke...