| Liver plays a critical role in the metabolisms of organism, such as glucose regulation, synthesis of many blood proteins, secretion of bile, biodegradation of toxic compounds, and others. Moreover the liver has an almost unlimited capacity to regenerate. So the research on characters of the liver proteins have an important significance in exploring the biological function of liver. Many proteins associated with the liver function are thermostable, such as: Hepatic Stimulator Substance(HSS), Augmentor of Liver Regeneration(ALR), Epidermal Growth Factor (EGF) et al. The objective of the project is to study the thermostable proteins of the mice liver, especially involved in the function and regeneration of liver. It was expected to find new thermostable proteins or new character of thermostablity. Then the crystal structure of the thermostable proteins would be determined by X-ray crystallography, and the relationship between structure and thermostability, structure and function would be studied.The innovative point of the project is that the thermostable proteins of the mice liver were studied for the first time. The new character of thermostability of glutathione peroxidase and regucalcin was discovered and studied, the structure and function of them was analzed through bioinformatic, and a certain foundation was built for analyzing the mechanism of the thermostability by X-ray crystallography and for protein engineering reform. The contents of the project research are as follow:1. Sample of mice liver was prepared and the thermostable proteins were separated by the technique of the Two-Dimensional Gel Electrophoresis(2-DE).2. Thermostable proteins were analyzed by MALDI-TOF-MS and bioinformatic.3. Thermostable proteins were tested(clone, expression and purification).4. Proteins' thermostability was detected.The results are as follow:1. According to the map, about fifty proteins were separated, in which five spots which molecular are in the range of 10-90kD and their separation effects better than others were selected to be processed by the technique of MALDI-TOF-MS. In term of the results, these five proteinswere as follow: glutathione peroxidase(GSH-Px), AF060087, nicotinate-nucleotide pyrophosphorylase, fumarylacetoacetate hydrolase and regucalcin. For the important significance of the glutathione peroxidase in the body and the regucalcin function related with liver regeneration, so they were cloned and expressed, and the thermostability was further determined.2. The results of two recombinant plasmids DNA were confirmed by digestion with restrictive endonuclease BamH I , Xho I and EcoR I , Xho I , and by DNA sequencing. The results indicated that GSH-Px and regucalcin cDNA were correctly inserted into the pGEX 4T-1 vector. Because of the selenocystein in the activated site is encoded by stop coden TGA, so we mutated it into TGC(encode cysteine) and expressed the whole GSH-Px.3. The GSH-Px fusion protein was induced and expressed by IPTG for 3 hours and the expression level of target protein was about 30 percent of the total bacterium proteins. After sonicated and centrifuged, the supernatant was purified through affinity chromatography: Glutathione Sepharose 4B, then the GST fusion protein was obtained. After digested by thrombin, SDS-PAGE showed two straps: one was GST and the other was GSH-Px. Regucalcin was expressed as inclusion body after induced by IPTG and the expression level of the target protein was about 30 percent of the total bacterium proteins.4. Detection of the thermostability: The mixture solution of GSH-Px digested by thrombin was treated at 65^0 for 30 minutes and centrifuged at 12000rpm for 20 minutes, the supernatant was collected and processed in SDS-PAGE, the result showed only one strap, proved that GSH-Px is thermostable. Moreover, GSH-Px was treated for 0, 5, 10, 20, 40 minutes at 65 掳C respectively, the thermostability was detected with the GSH-Px Activity Test Kit, and the result showed that the activity of GSH-Px didn't change too much, fur... |
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