| Title |
Expression, Purification, and Characterization of Recombinant Fibulin-5 in a Prokaryote Expression System |
| Author |
Myoung Seok Jeong1, Chang Soo Kang1, Yeon Soo Han2, and In Seok Bang1* |
| Address |
1Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University, Asan 336-795, Republic of Korea, 2Department Agricultural Biology, Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, Republic of Korea |
| Bibliography |
Journal of Microbiology, 48(5),695-700, 2010,
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| DOI |
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| Key Words |
fibulins, anti-angiogenesis, CAM assay, fusion protein, prokaryotic expression, protein purification |
| Abstract |
Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N2+-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L. |
