Hepatocyte growth factor (HGF) is a copper-binding protein: a facile probe 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-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.
Rahimi
N
N
Department of Pathology, Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Etchells
S
S
Elliott
B
B
D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D004247:DNA / Q000096:biosynthesis; D017228:Hepatocyte Growth Factor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification
D008024:Ligands; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D003300:Copper; D004247:DNA