HEPATOCYTE GROWTH-FACTOR (HGF) IS A COPPER-BINDING PROTEIN - A FACILEPROBE FOR PURIFICATION OF HGF BY IMMOBILIZED CU(II)-AFFINITY CHROMATOGRAPHY

Hepatocyte growth factor (HGF) is a multifunctional protein expressed in a variety of cell types and tissues. Here we describe a novel one-s tep method to separate and identify HGF, based on a unique interaction between HGF and Cu(II). Conditioned medium (CM) from mouse 3T3-L1 adi pocytes which contains HGF or purified human recombinant HGF was used for analysis. Mouse 3T3-L1 adipocyte CM was applied to a Cu(II)-affini ty column and rinsed with equilibration buffer. HGF was then eluted wi th 10 mM imidazole. Fractions eluted from the column were analyzed by SDS-PAGE. Analysis by silver staining revealed an 85-kDa protein. Furt her analysis by Western blotting with polyclonal anti-HGF IgG demonstr ated that this protein corresponded to HGF. Human recombinant HGF, whe n applied to a Cu(II)-affinity column, showed a stronger affinity to C u(II) than did mouse HGF. Human recombinant HGF was not eluted from th e Cu(II) column with either 10 or 20 mM imidazole; however, it was rea dily eluted with 40 mM imidazole. The percentages of recovery of both human and mouse HGF were greater than 90%, Both mouse HGF and human re combinant HGF eluted from the Cu(II)-affinity column retained their bi ological activity as measured by HGF-induced cell proliferation of Mv1 Lu cells. Our findings provide the first evidence that HGF is a copper -binding protein and that a Cu(II)-affinity column can be used for eff icient one-step purification of biologically active HGF. (C) 1996 Acad emic Press, Inc.