Expression Of Osteopontin RGD Peptide In E.coli And Its Biological Activities

Objective: The formation of neointima induced by endothelium injury is an important cause of percutaneous transluminal coronary angioplasty(PTCA). When the vessels are injured, vascular smooth muscle cells (VSMC) in media which are stimulated by growth factors and cytokines reverse from the contractile (differentiated) state to the synthetic (dedifferentiated) state, and migrate into endothelium and release cytokines and extracellular matrix (ECM) proteins. Migration of VSMC is regulated by the interaction of OPN with integrinαvβ3 on surface of cells. OPN plays a key role in VSMC adhesion and migration by interactions of RGD motif in OPN withαvβ3 andαvβ5. In addition, the SVVYGLR sequence of OPN regulates many cell behaviors by interactions withα9β1 andα4β1. Previous studies indicated that peptides containing a RGD sequence and peptides with a SVVYGLR sequence blocked the binding of OPN to the integrins and inhibited VSMC adhesion, migration, and macrophage infiltration. Small peptides that display inhibitory roles as decoy oligopeptides may be used to study the function of proteins. This study constructed prokaryotic expression plasmids containing RGD and SVVYGLR cDNA sequences. E. coli transformed with the construct could be induced to express His-RGD fusion protein.Methods:1 Construction of recombinant plasmidsHis-RGD prokaryotic expression plasmid was constructed by cloning synthesized OPN 13 peptide-coding sequence into prokaryotic expression vector pET-32c(+). The recombinant plasmid was identified by DNA sequence determination.2 Expression of His-RGD fusion protein in E.coli2.1 Expression of His-RGD fusion proteinE.coli BL21(DE3) transformed by the construct was induced by IPTG under different conditions. Supernatant and precipitation of pET-32c-RGD transformant lysates were analyzed by SDS-PAGE.2.2 Purification of His-RGD fusion proteinThe fusion proteins were purified from the supernatants of the culture lysates with Ni-NTA His Bind Resin metal chelation chromatography. 25 mg of purified soluble His-RGD fusion protein was obtained from 100 ml bacteria culture.3 Detection of biological activities of His-RGD fusion protein3.1 Adhesion assayVSMCs suspended in 1% NCS medium were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h, and then the cells were seeded in 96-well plate coated by BSA (20 mg/L) or OPN (20 mg/L). The number of the adhesion cells per well was detected, which represented the activity of the cell adhesion. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls.3.2 Wound healing assayVSMCs grown to 100% confluent monolayers were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h,and then scratched to form a wound. After addition of OPN (20 mg/L) or BSA (20 mg/L) for 24 h, wound healing was observed by microscope, which represented the activity of the cell migration. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls to identify the specificity of migration inhibition.Results:1 Construction and identification of recombinant plasmids pET-32c-RGD.The recombinant plasmids were identified by NcoⅠ/BamH I digestion. DNA sequence determination and restriction enzyme analysis showed that the inserted fragment of pET-32c-RGD was correct.2 Expression and purification of His-RGD fusion proteinHis-RGD fusion proteins were effectively expressed in E.coli transformed by pET-32c-RGD after induction with 10μ mol/L IPTG for 7 h at 25℃.The fusion proteins were purified by Ni-NTA His Bind Resin metal chelation chromatography. The final yield of His-RGD fusion proteins was 25 mg/100 ml culture, and the purity of His-RGD fusion protein was over 95%.3 Effect of His-RGD fusion protein on VSMC adhesion and migrationThe adhesion assay indicated that His-RGD fusion protein dose-dependently decreased the adhesion and migration of VSMC, the inhibitiory effect reached the peak after preincubation by 450 mg/ml OPN-13-peptide.Conclusions:1 Prokaryotic expression plasmid pET-32c-RGD is successfully constructed.2 His-RGD fusion protein is effectively expressed in E.coli after IPTG induction.3 His-RGD fusion protein is purified with Ni-NTA His Bind Resin metal chelation chromatography. The yield of purified His-RGD fusion proteins is 25 mg/100 ml culture, and the purity of fusion protein is over 95%.4 His-RGD fusion protein can dose-dependently decrease adhesion and migration of VSMC.