Fig. 1

Expression and purification of RGD-p21Ras-scFv recombinant antibody in E.coil system. (A)The recombinant expression plasmids pET32a-RRS were set up by inserting the RGD-p21Ras-scFv gene into the corresponding enzyme cleavage site of the plasmid vector. (B) PCR showed the RGD-p21Ras-scFv gene was successfully transferred into pET32a plasmid. (C) The highest expression of RGD-p21Ras-scFv was detected in Origami(DE3) and Origami B(DE3) inclusion bodies by SDS-PAGE. The red arrow showed the target protein. (D) The RGD-p21Ras-scFv was expression in different concentration of IPTG from 0.2mM to 1.6mM. The red arrow shows the optimum inducible expression conditions. (E) After the suitable concentration of IPTG was chosen(0.6mM), the induced time was investigated from 4 to 20 h. The red arrow shows the optimum inducible expression conditions. (F) SDS-PAGE showed theRGD-p21Ras-scFv was more highly expressed in the self-induction system than in IPTG as detected by SDS-PAGE analysis. The red arrow shows the target protein. (G) Affinity chromatogram (left) and SDS-PAGE (right) of RGD-p21Ras-scFv recombinant antibody from Ni2 + affinity resin. The wash fraction contains 20 mM imidazole, and the elution fraction contains 250 mM imidazole. (H) RGD-p21Ras-scFv recombinant antibody was more highly expressed in inclusion bodies than in soluble proteins as detected by SDS-PAGE analysis. (I) Ion-exchange chromatography of RGD-p21Ras-scFv from DEAE resin (left) and SDS-PAGE (right). The refolding protein to loading up after adjusted the pH at 8.0 and the elution fraction with 0.3 mol/L NaCl in elution buffer. (J) Comparison of the wet weight of bacteria expressed in small scale shaker in laboratory and fermentation. Fermentation can significantly increase the yield (cell wet weight)