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Re-identification of Osteopontin from Bovine Milk by Its Inhibition for the Growth of Calcium Oxalate Crystal in Vitro and Its Secondary Structure

Author: ShiJie
Tutor: LiuNing
School: Northeast Agricultural University
Course: Of Food Science
Keywords: Bovine milk OPN Purification process optimizatin Re-Identification Secondary structure determination
CLC: TQ464.7
Type: Master's thesis
Year: 2010
Downloads: 19
Quote: 0
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Abstract


Osteopontin(OPN) is a phosphorylated acidic glycoprotein, and exists in mammalian tissue and body fluids. OPN can induce the mature of the mineralized bone matrix, regulate the formation of hydroxyapatite, promote the reconstruction of mineralized tissues, as well as prevent the decline in bone density. We found that the purified OPN from bovine milk was slightly different from the Protein Data Bank(PDB) when we took the research on how it promoted the reconstruction processing of bone. Therefore, this study aims to identify the purified OPN using the the biological characteristics of OPN which can inhibit the growth of calcium oxalate crystals.The results of OPN inhibited the growth of calcium oxalate crystals is consistent with the SDS gel electrophoresis results, demonstrates that the purified protein has the characteristics of OPN. Thererof this study gives the basis for the research on the relationship between OPN and bone metabolism.The secondary structure analyzing of OPN from bovine milk using the application of existing instruments at present is still blank, so far we still can not give its precise structure through determination. In this study, Fourier Transform Infrared Spectroscopy were used to concretely analysis the secondary structure of purified OPN, which can give the helix, corner and so on of each conformation that exit in the secondary structure of OPN. And this has important guiding significance on the study of the relationship between structure and function of OPN.The study of this subject mainly consists of 4 parts. Firstly, isolated the OPN from bovine milk through reversed-phase chromatography, as well as removed of casein and other purification process optimizatins. Secondly, analysised the chromatography eluting fractions which used SDS-PAGE, re-qualified the various chromatographic elution peaks according to the role of OPN on calcium oxalate crystal growth inhibition activity. Thirdly, assayed the OPN content of each Elution peaks from various purification steps, and then, made the final production(OPN) purity determination. Fourthly, after freeze-drying of the purified samples, its secondary structure was determined by Fourier transform infrared spectroscopy determination. The results of this research are as follows:⒈The result of purification process optimizatins and re-identification was showed, after the treatment of the removal of casein between the DEAE-Sephacel ion-exchange chromatography and Phenyl-Sepharose Hydrophobic Chromatography, at the end of Hydrophobic Interaction Chromatography we obtained homogeneous component of OPN, which can be well confirmed by conventional SDS-PAGE bands. Finally, according to OPN inhibition biological activity on the growth of calcium oxalate crystals, the two elution fractions through the SOURCE 5RPC ST RP-interaction chromatography were identified both had crystal growth inhibitory activity, the two fractions were inferred as the different molecular fragments for the OPN respectively. Further, in accordance with the calcium ion concentration changes of supernatant in which calcium oxalate crystal growned, the dose-response curve of OPN on calcium oxalate crystal growth inhibition was quantitatively determined by atomic absorption spectrometry, confirmed when the crystal growth inhibition rate reached 50﹪, the requirement concentration for the purified OPN was about 24 nM.⒉According to Bradford assay measurement of the total protein content, as well as ELISA determination of the OPN content, can obtaine 8.98 mg OPN from 1 L of raw bovine milk after optimized purification steps, the purity was 0.556 mg/mg protein.⒊Fourier transform infrared spectroscopy was used for secondary structure determination on OPN freeze-dried powder which purified from bovine milk, combined with deconvolution and second derivative fitting processing, analisised purified bovine milk OPN secondary structure contained approximatelyα-helix: 70﹪;β-folding: 30﹪.The results proved that it can be well identified where the target protein(OPN)is during each purification step based on the inhibit activity of OPN on the crystal growth combined with electrophoresis. This study not only provid raw material purification technology for the in vitro study on its activity and parameters as well as technical support for the industrial extraction of bovine milk OPN, but also make it possible for OPN to be added to a variety of health foods and drugs as a function factor to the prevention and treatment for related symptoms. Study on the secondary structure of the OPN can lay the foundation for its spatial structure, at the same time provide the basis for the study on its structure-function relationships.

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CLC: > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Drug production of biological products > Amino acids,peptides,proteins
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