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| Cloning, Expression, Purification and its Biological Activity Study of the Soluble Tripolymer Recombinant Angiogenesis Inhibitor Kringle 5 |
| MIAO Liang, XU Li-hua, JI Xu, BIAN Liu-jiao |
| College of Life Sciences, Northwest University, Xi’an 710069, China |
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Abstract The Kringle 5 domain of plasminogen, which was previously shown to inhibit angiogenesis in vitro and vivo, is one of the most potent angiogenesis inhibitors described to date.The Kringle 5 gene was amplified from pET32a-K5 by PCR, and then was inserted into pET15b to construct a prokaryotic vector pET15b-k5.The target protein was highly expressed in E.coli BL21(DE3) and was mainly present in the precipitation observed by SDS-PAGE. The protein was purified by ion-exchange chromatography (SP Sepharose Fast-Flow) using the NaCl linear gradient strategy. The analysis results of SDS-PAGE showed that the purity of obtained Kringle 5 was about 98%. After desalting with size-exclusion chromatography, it was found that it inhibited the blood vessel growth of chick embryo chorioallantoic membrane effectively. This simple, effective method may be applied in the future in mass production of Kringle 5 and lay a solid base for detailed evaluation of bioactivities of Kringle 5.
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Received: 10 November 2010
Published: 01 April 2011
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