High level expression of kringle 5 fragment of plasminogen in Pichia pastoris.
Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300 mgl(-1). Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.
Zhou
Yufei
Y
National Key Laboratory of Protein Engineering and Plant Gene Engineering, LSC, Peking University, Beijing, PR China.
Zheng
Quan
Q
Gao
Jin
J
Gu
Jun
J
D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D018082:Kringles / Q000235:genetics*; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015003:Yeasts / Q000235:genetics
D011994:Recombinant Proteins; D010958:Plasminogen