EXPRESSION OF PLATELET-DERIVED ENDOTHELIAL-CELL GROWTH-FACTOR IN ESCHERICHIA-COLI AND CONFIRMATION OF ITS THYMIDINE PHOSPHORYLASE-ACTIVITY

Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [H-3]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S.I. (1992) Nature 356,668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [H-3] methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.