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Expression of Osteopontin in E.coli and Its Biological Activities

Author: ZuoChao
Tutor: WenJinKun
School: Hebei Medical University
Course: Biochemistry and Molecular Biology
Keywords: Osteopontin GST-OPN fusion protein Prokaryotic cell expression Protein Purification Biological activity
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 88
Quote: 0
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Abstract


Objective: Osteopontin (Osteopontin, OPN) is an important function in the extracellular matrix protein, by the synthesis and secretion of a variety of tissue cells. In recent years, studies have shown that OPN as airframe reactive protein, inflammation in the body, the organization has an important function and role in the pathological process of damage and repair. Through integration with the surface of vascular endothelial cells and smooth muscle cells (VSMC) in the cardiovascular system, OPN receptor interactions mediated cell adhesion and migration, and thus participate in the occurrence and development process of the vascular endothelial injury caused by intimal hyperplasia. In order to study the molecular mechanisms of OPN-induced VSMC adhesion and migration and its role in the intimal hyperplasia preparation OPN is necessary. To this end, this study, the use of recombinant DNA technology, restructuring OPN cDNA and prokaryotic expression vector carrying the coding sequence of GST, the GST-OPN fusion protein expressed in E. coli for further study of the protein in the intimal hyperplasia lays the foundation. Methods: 1 OPN original expression plasmid of building OPN cDNA fragment was cloned into the pGEX-4T-1 original expression vector construct pGEX-4T-1-OPN plasmid, restriction endonuclease map and DNA sequence analysis of recombinant plasmids carried identification . 2 GST-OPN fusion protein expression in E. coli 2.1 GST-OPN fusion protein induced expression of the transformed E. coli, pGEX-4T-1-OPN, the IPTG concentration, induction time and induction temperature filter optimization, to determine the GST- OPN fusion protein induced expression of the optimum conditions. PGEX-4T-1-OPN transforming bacteria after induction culture under optimum conditions, the cell lysate after centrifugation, the supernatant and pellet were collected and checked by SDS-PAGE, the existence form of the fusion protein in cells. 2.2 GST-OPN fusion protein isolated and purified bacterial supernatant after lysis and centrifugation, the GST-OPN fusion proteins were purified with GST-Sepharose 4B affinity chromatography. 3 GST-OPN fusion protein biological activity the 3.1 cell adhesion test passaged VSMC were seeded to 96-well plate with OPN packet is detected, the detection unit time adhesion to the number of cells in the orifice plate, as an expression of the cell adhesion activity. Natural OPN and GST protein extracted from VSMC medium as parallel control, to determine the GST-OPN to promote cell adhesion specificity. 3.2 wound healing experiments cell passaging, the VSMC were inoculated in the orifice plate with the slides, and the cells were grown to 100% confluence, the suction head on slide with sterile scratches. PBS wash scraped cells, slides back into the well plate, each well was added to 23 mg / L of GST-OPN continue after 24 h of incubation, the cells were observed at low magnification wound healing degree of, as an expression of the cell migration activity. Natural OPN and GST protein extracted from VSMC medium as parallel control to determine the GST-OPN to promote the specificity of cell migration. Results: a recombinant plasmid pGEX-4T-1-OPN build as a template for PCR amplification to obtain a recombinant plasmid containing the cDNA sequence of OPN OP10 (Dr.Larry Fisher kindly) OPN cDNA fragment ligated with pGEX-4T-1 vector The recombinant plasmid. Electrophoretic identification of recombinant plasmid after Bam HI / Xho Ⅰ double digestion length of about 860 bp insert fragment, consistent with the expected results. OPN cDNA sequencing analysis in GenBank sequences were compared, prove that the insertion fragment sequence is correct. 2 GST-OPN fusion protein induced expression host strain transformed pGEX-4T-1-OPN molecular weight of approximately 75 kD protein can be expressed after IPTG induction. Culture OD600 of = 1.5 at a ratio of 1:50 inoculated in LB medium containing 0.1 mmol / L IPTG, 25 ° C induced culture for 6 h under the conditions, the expression amount of the GST-of OPN fusion protein in the bacterial protein The largest proportion of the fusion protein in soluble form. This condition as a GST-OPN fusion proteins induce expression of the best conditions. 3 GST-OPN fusion protein isolated and purified into the pGEX-4T-1-OPN host bacterium under the best conditions, the induction culture, the cells were collected by centrifugation, resuspended in PBS (pH 7.3) after the addition of NaOH adjusted to pH 8.0, in order to achieve the lysozyme play a role in the optimum pH, digestion with lysozyme room temperature, add Triton X-100 in order to prevent protein crosslinking. Collected by centrifugation and the supernatant was added glutathione agarose beads incubated, washed with PBS after 4 to 5 times, with glutathione elution with several elution merge collected eluant. Take appropriate eluate was subjected to SDS-PAGE, the results to be seen clearly, a single 75 kD protein bands described by affinity chromatography and the purified GST-OPN reached electrophoresis pure. 100 ml culture to obtain about 3 mg of soluble GST-OPN fusion protein was purified by affinity chromatography and after purification. 4 GST-OPN fusion protein of VSMC adhesion observed in this study recombinant GST-OPN fusion protein on the cell adhesion. It was found that the GST-OPN fusion of the protein can significantly promote VSMC adhesion, its role was no significant difference with natural OPN. The parallel control GST protein does not affect VSMC adhesion. 5 GST-OPN fusion protein of VSMC migration VSMC migration by the interaction of cells with the extracellular matrix, and a variety of cytokines mediated. Disintegrin as the primary ligand, the extracellular matrix of OPN to play an important role in the VSMC migration. This experimental study recombinant OPN on cell migration, adhesion assay grouping. GST-OPN can significantly promote cell migration, its role was no significant difference with natural OPN, parallel control experiments GST protein did not affect VSMC migration. Conclusion: successfully constructed the pGEX-4T-1-OPN prokaryotic expression plasmid containing the OPN gene sequence. After IPTG concentration, induction temperature, induction time conditions optimized under the conditions of 25 ° C with 0.1 mmol / L IPTG induction 6 h, GST-OPN fusion of the protein to be effectively expressed in E. coli. 3 bacterial supernatant after lysis and centrifuged to obtain electrophoretically pure GST-OPN fusion protein after GST-Sepharose 4B affinity chromatography. 4 GST-OPN fusion protein can significantly promote VSMC adhesion. 5 GST-OPN fusion protein can significantly promote VSMC migration.

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