Production and purification of active FGF2 via recombinant fusion protein.
Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.
Lemaître
G
G
Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, URA-CNRS 1813, Université Paris Val de Marne, Créteil, France.
Laaroubi
K
K
Soulet
L
L
Barritault
D
D
Miskulin
M
M
D000688:Amylose / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology
D002352:Carrier Proteins; D018076:DNA, Complementary; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D000688:Amylose