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 [3H]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 [3H]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.
Moghaddam
A
A
Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, U.K.
Bicknell
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004926:Escherichia coli; D005285:Fermentation; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D011994:Recombinant Proteins; C012439:methylthymidine; D013939:Thymidine Phosphorylase; D013936:Thymidine