| Selenium, as an essential trace element in organism, mainly functionsas the constituent of selenoenzymes. Glutathione peroxidase (GPX) isa well-known selenoenzyme that works as an antioxidant and catalyzesthe reduction of harmful peroxide by glutathione and protects cellsagainst oxidative damage. Because many diseases are related tooxidative stress, GPX is an ancient foe of many diseases. Antioxidantsare very useful for biological bodies, and considerable effort has beenspent to find compounds that could imitate the properties of GPX.Although many GPX mimics have been made, they possess seriousdisadvantages: low activity, low solubility in water, and, in somecases, toxicity. In order to overcome these drawbacks, we have proposeda new strategy of imitating GPX. First, a receptor with a substratebinding site is generated. Next, a catalytic group is incorporated intothe receptor near the substrate binding site, allowing the catalyticgroup access to the functional group of the substrate, and finally ahighly efficient enzyme mimic is obtained.1. Chemical chaperones increase the expression level ofsoluble single-chain Fv antibody (scFv2F3)The selenium-containing single chain Fv fragment of monoclonalantibody 2F3 (Se-scFv2F3) is a high efficient GPX mimic prepared withthe technology of preparation monoclonal antibody and recombinant DNA.It has more important prospects for application in clinic than thoseearly developed because of its high activity and low antigenicity.Se-scFv2F3 is generated by chemical modification of the single chainFv fragment of monoclonal antibody 2F3 (scFv2F3) and the scFv2F3 waslargely expressed in inclusion form in E. coli. Because scFv2F3 ininclusion form cannot be used directly to prepare Se-scFv2F3 beforeits renaturation and the efficiency of renaturation is low, we havebegun to express scFv2F3 as a soluble form in the periplasm of hostusing a new expression vector. Although scFv2F3 was found in the solublefraction, allowing purification under native nondenaturing conditions,a significant quantity of scFv2F3 was stored in inclusion body.To overcome the formation of aggregates, and the subsequent packaginginto inclusion bodies during in vitro folding, chemical chaperoneshave been used. Chemical chaperones are low molecular weight compounds,which can stabilize the structure of proteins in vitro. These reagentsinclude the organic solvent dimethyl sulfoxide and cellular osmolytesglycerol, glucose and trimethylamine N-oxide. A lot of reports havebeen shown that glycerol, as a chemical chaperones, can stabilize theprotein conformation, correct its defective folding and facilitate itsswitch from inclusion bodies to the soluble form. Cyclodextrins (CDs)are cyclic oligosaccharides with a hydrophilic outer surface and asomewhat lipophilic central cavity;it has been used to prevent theirreversible aggregation of partially refolded proteins.As mentioned above, glycerol and glucose added to the culture mediumfacilitate the expression of some soluble recombination proteins. Inthis work, we investigated whether they have the similar effects onthe expression of soluble scFv2F3. Besides, the examples concerningCDs mentioned above indicated the effects of CDs on protein refoldingin vitro.We studied the effects of chemical chaperones, such as glycerol,glucose and β-cyclodextrin added to the culture medium, on expressionof soluble form of scFv2F3. The expression level was evaluated bydetermining the content of scFv2F3 in whole cell soluble proteins. Ourresults suggest that glycerol and β-cyclodextrin greatly increase theexpression, and β-cyclodextrin is more effective compared withglycerol. Glucose has a slight effect on the expression. To ourknowledge, this is the first example that β-cyclodextrin was used asa chemical chaperone during cell culture to improve the expressionlevel of recombinant protein. In addition, glucose and β-cyclodextrincan decrease the toxic effect of IPTG to cells.2. Construction of selenium-containing 5-mer peptidesWe design and obtain a kind of short peptide identifying GSH withsolid-phase synthesis technology based on the active center of nativeGPX. This mimic is characterized with HPLC-MS. Its activity is 11U/μmol according to Wilson's methods and it has the highest activityrelative to its molecular weight compared with other small molecularmimics. The optimal pH and temperature of Se-5P catalyzing thereduction of H2O2 by GSH are found to be 8.94 and 43.2 ℃ respectively.In addition, Se-5P shows similar kinetic behavior as natural GPXs, thatis they both abide the Ping-Pong mechanism. And the values of kcat/KmH2O2and kcat/KmGSH of Se-5P are higher compared with natural enzyme, indicatingthat Se-5P has higher affinity with both substrates. In summary, wehave successfully synthesized a GPX mimic with small size, highactivity and specific binding site for substrates. These advantagesmade the novel mimic have very extensive perspective for use in medicineand drugs.3. The biological effects of of selenium-containing5-mer peptidesWe construct the Vc/Fe2+ ROS-induced Mitochondria and H2O2-inducedculture hepatocyte cells damage model systems, demonstrating thedamaged mitochondria and hepatocyte cells have great changes in thetwo systems. The damages include: the extent of mitochondria swellingis increased, lipid peroxidation is occurred with dose-dependent,penetration of the cell membrane decrease, DNA in the cellsfragmentations and the cell viability decreases. We investigate theprotective effects of mitochondria and cultured hepatocyte cells bySe-5P using the two damage systems, indicating that they can inhibitthe swelling of mitochondria, the increase of the MDA content, and thedecrease of the cell viability. They also can prevent the leakage ofLDH to sustain the well penetration in cell membrane. These resultsindicate that Se-5P can scavenge free radicals to sustain the functionof antioxidative defense system in cells.In addition, we found that Se-5P cannot be inactivated by freeradicals induced by H2O2 while the native GPX was inactivated by freeradicals, indicating that Se-5P is superior to the native GPX inthis aspect. H2O2-induced culture hepatocyte cells damage is similar tooxidative stress occurred in the organism, thus, the studies forthe Se-5P using the two systems provide the theory bases for thisGPX mimic to treat the diseases induced by free radicals, such ascataract, angiocardiopathy and cancers.
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