Expression of kringle 5 domain of human plasminogen in Pichia pastoris.
The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.
Zhu
Ming
M
State Key Laboratory of Pharmaceutical Biotechnology, Institute of Molecular and Cell Biology and Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing, P.R. China.
Cheng
Jian
J
Hua
Zi-Chun
ZC
D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects; D015870:Gene Expression; D016681:Genome, Fungal; D006801:Humans; D018082:Kringles / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D010958:Plasminogen / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA