Expression Of Osteopontin In E.coli And Its Biological Activities

Objective: Wound healing is a complicated process. In the early stages of injury, leukocyte and platelets activated by contact with injured endothelium release a series of cytokines and proinflammatory mediators that can stimulate angiogenesis and fibrous tissue proliferation. The formation of new vessels is an important event of wound recovery. We found that as an important signal molecule, OPN can stimulate wound healing by inducing new endothelium proliferation and VSMC ( vascular smooth muscle cell ) migration. In this study,we constructed prokaryotic expression plasmids containing OPN gene sequences by recombinant DNA technology. Transformed E. coli could be induced to express GST-OPN fusion protein.Methods:1 Construction of recombinant plasmids pGEX-4T-1-OPN plasmid was constructed by cloning OPN cDNA into pGEX-4T-1 vector.2 Expression of GST-OPN fusion protein in E.coli2.1 The optimal condition of expression of GST-OPN fusion proteinTransformed E.coli was induced by different IPTG concentrations, times and temperature to identify the optimal induction condition. Supernatant and precipitates of pGEX-4T-1-OPN transformant lysates were analyzed by SDS-PAGE to observe the position of fusion protein in the cells.2.2 Purification of GST-OPN fusion proteinThe supernatant was collected by centrifugation and purified via GST-Sepharose 4B affinity resin.3 Detection of biological activities of GST-OPN fusion protein3.1 Adhesion assayVSMCs were suspended in 1 % NCS medium, and then, the cells were seeded in 96-well plate coated by OPN. The number of the adhesion cells per well was detected, which represented the activity of the cell adhesion. The nature OPN and GST protein were used to serve as parallel controls to identify the specificity of cell adhesion.3.2 Wound healing assayVSMCs were seeded in the plate with glass slide. After the cells were grown to 100 % confluent monolayers, the cells were scratched to form a wound, and then, stimulated with OPN for 24 h, wound healing was observed by microscope, which represented the activity of the cell migration. The nature OPN and GST protein were used to serve as parallel controls to identify the specificity of cell migration.Results:1 Construction of recombinant plasmid pGEX-4T-1-OPN The analysis of DNA sequence and restriction enzyme analysis showed that the inserted fragments in pGEX-4T-1-OPN were consistent with anticipated results.2 Expression of GST-OPN fusion proteinGST-OPN fusion protein could be expressed in E. coli transformed by pGEX-4T-1-OPN after induction, which prominently existed in supernatant. The expression of GST-OPN fusion protein was the highest under condition induced by 0.1 mmol/L IPTG for 6 h.3 Purification of GST-OPN fusion proteinThe fusion protein was purified via GST-Sepharose 4B affinity resin, 3 mg of purified GST-OPN fusion protein was obtained in 100 ml culture, the purity of GST-OPN fusion protein was over 95%.4 Effect of GST-OPN fusion protein on VSMC adhesionThe result of adhesion assay indicated that GST-OPN fusion protein could increase adhesion of VSMC, and this effect was similar to that of nature OPN. GST protein didn't affect cell adhesion.5 Effect of GST-OPN fusion protein on VSMC migrationThe result of wound healing assay showed that GST-OPN fusion protein could promote migration of VSMC, and this effect was similar to that of nature OPN. GST protein didn't affect cell migration.Conclusions: 1 Prokaryotic expression plasmid pGEX-4T-1-OPN is successfully constructed.2 GST-OPN fusion protein is effectively expressed in E.coli under some condition.3 GST-OPN fusion protein is purified via GST-Sepharose 4B affinity resin, and the purity of fusion protein was over 95%.4 GST-OPN fusion protein can increase adhesion of VSMC.5 GST-OPN fusion protein can promote migration of VSMC.