Expression of biologically active kringle 5 domain of human plasminogen in Escherichia coli.
The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelial cell specifically, and shows promise in anti-angiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When previously expressed in Escherichia coli, recombinant kringle 5 mainly deposited as inactive, insoluble inclusion bodies and the refolding yield was low. In the present study, human kringle 5 was fusion-expressed with GST (gluthathione-S-transferase) under the control of T7 promoter in E. coli. The IPTG-induced GST-kringle 5 was about 20% of the total cellular proteins and, among the expressed GST-kringle 5 proteins, 80% was present in the supernatant. The GST-kringle 5 fusion protein exhibited some anti-proliferation activity towards bovine capillary endothelial cells. After GST-kringle 5 purification, subsequent enterokinase release of intact kringle 5 from the fusion protein and further purification by gluthathione-Sepharose 4B affinity chromatography, the recombinant kringle 5, with a yield of 10.5 mg/L culture, displayed apparent inhibition of endothelial cell proliferation in a dose-dependent manner with ED50 about 20 nM.
Zhang
Hong-Xia
HX
State Key Laboratory of Pharmaceutical Biotechnology, Institute of Molecular and Cell Biology, PR China.
Fang
Lei
L
Cheng
Jian
J
Wei
Zheng
Z
Hua
Zi-Chun
ZC
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010958:Plasminogen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C491499:plasminogen kringle 5; D010958:Plasminogen