EXPRESSION OF PLATELET-DERIVED ENDOTHELIAL-CELL GROWTH-FACTOR IN ESCHERICHIA-COLI AND CONFIRMATION OF ITS THYMIDINE PHOSPHORYLASE-ACTIVITY
EXPRESSION OF PLATELET-DERIVED ENDOTHELIAL-CELL GROWTH-FACTOR IN ESCHERICHIA-COLI AND CONFIRMATION OF ITS THYMIDINE PHOSPHORYLASE-ACTIVITY
复制标题
DOI：
10.1021/bi00163a024
复制
发表时间：
1992-12-08
期刊：
影响因子：
2.9
通讯作者：
BICKNELL, R
中科院分区：
文献类型：
--
作者：
MOGHADDAM, A;BICKNELL, R
血小板源性内皮细胞生长因子（PD-ECGF）已在大肠杆菌中表达为谷胱甘肽S-转移酶（GST）融合蛋白。融合蛋白通过谷胱甘肽-琼脂糖珠上的一步亲和层析纯化，并且重组PD-ECGF用凝血酶从其GST前导肽上蛋白水解切割以产生纯蛋白。重组PD-ECGF刺激[H-3]甲基胸苷摄取内皮细胞在体外，但是，我们无法检测到刺激细胞增殖的各种条件下。我们证实，雅阁最近报道的PD-ECGF和人胸苷磷酸化酶是相同基因的产物[Furukawa，T.，Yoshimura，A.，Sumizawa，T.，Haraguchi，M.，& Akiyama，S.I.（1992）Nature 356，668]重组PD-ECGF具有与大肠杆菌相当的胸苷磷酸化酶活性。大肠杆菌胸苷磷酸化酶。进一步，E.在[H-3]甲基胸苷摄取试验中，大肠杆菌胸苷磷酸化酶能够模拟重组PD-ECGF的活性，并且重组PD-ECGF对内皮细胞摄取胸苷的作用似乎是由于调节细胞胸苷库。PD-ECGF刺激血管生成的机制仍有待阐明。
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.