Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system.
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 N(2+)-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.
Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University, Asan 336-795, Republic of Korea.
Myoung Seok
MS
Jeong
Chang Soo
CS
Kang
Yeon Soo
YS
Han
In Seok
IS
Bang
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D042783:Endothelial Cells / Q000187:drug effects; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; C492373:FBLN5 protein, human; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase