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  • Title: Hepatocyte growth factor (HGF) is a copper-binding protein: a facile probe for purification of HGF by immobilized Cu(II)-affinity chromatography.
    Author: Rahimi N, Etchells S, Elliott B.
    Journal: Protein Expr Purif; 1996 May; 7(3):329-33. PubMed ID: 8860660.
    Abstract:
    Hepatocyte growth factor (HGF) is a multifunctional protein expressed in a variety of cell types and tissues. Here we describe a novel one-step method to separate and identify HGF, based on a unique interaction between HGF and Cu(II). Conditioned medium (CM) from mouse 3T3-L1 adipocytes which contains HGF or purified human recombinant HGF was used for analysis. Mouse 3T3-L1 adipocyte CM was applied to a Cu(II)-affinity column and rinsed with equilibration buffer. HGF was then eluted with 10 mM imidazole. Fractions eluted from the column were analyzed by SDS-PAGE. Analysis by silver staining revealed an 85kDa protein. Further analysis by Western blotting with polyclonal anti-HGF IgG demonstrated that this protein corresponded to HGF. Human recombinant HGF, when applied to a Cu(II)-affinity column, showed a stronger affinity to Cu(II) than did mouse HGF. Human recombinant HGF was not eluted from the Cu(II) column with either 10 or 20 mM imidazole; however, it was readily eluted with 40 mM imidazole. The percentages of recovery of both human and mouse HGF were greater than 90%. Both mouse HGF and human recombinant HGF eluted from the Cu(II)-affinity column retained their biological activity as measured by HGF-induced cell proliferation of Mv1Lu 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 efficient one-step purification of biologically active HGF.
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