Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected...

A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by itself, did not have any detectable biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful production of bioactive recombinant HGF in large quantities, which will allow purification on the milligram scale of pro-HGF and will permit future studies to elucidate pathways involved in HGF activation by its target tissues.