Solubilization and purification of <i>Escherichia coli</i> expressed GST-fusion human vascular endothelial growth factors with <i>N-Lauroylsarcosine</i>

Abstract

Vascular endothelial growth factor (VEGF) is a potent mitogen for tumor angiogenesis. Clinically, VEGF detection in human blood can be expected to be used in the very near future for cancer screening, prognosis, monitoring of therapy and diagnosis. VEGF has been identified as the target for the treatment of cancer. Though prokaryotic expression of VEGF has been done, the solubilization and purification is time consuming and empirical. In this study, VEGF165 and VEGF121 were cloned into
pGEX-4T-1 vector, and GST-VEGF fusion proteins were expressed in Escherichia coli at 37°C. The inclusion bodies of GST-VEGF fusion proteins were solubilized with N-Lauroylsarcosine (sarkosyl). Briefly, the cell suspension with inclusion body was added with sarkosyl at a final concentration of 1.5%. After the disruption of cells, the clarified supernatant containing sarkosyl was added with Triton X-100 at a final concentration of 3%. The GST-VEGFs were purified by affinity chromatography on glutathione Sepharose 4B. The overall yield was approximately 10 – 12 mg/l cell culture. The binding assay showed that the GST-VEGF165 binds to VEGF receptor in a dose dependent manner. The current work provides a novel procedure for solubilization and purification of GST-VEGF fusion proteins, and no laborious procedures for separation of inclusion bodies and renaturation were needed.