EXPRESSION AND CHARACTERIZATION OF BIOLOGICALLY-ACTIVE HUMAN HEPATOCYTE GROWTH-FACTOR (HGF) BY INSECT CELLS INFECTED WITH HGF-RECOMBINANT BACULOVIRUS

A cDNA containing the entire coding sequence of human hepatocyte growt h factor (HGF) [also known as scatter factor (SF)] was inserted into t he genome of Autographa california nuclear polyhedrosis virus (baculov irus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant vi rus secrete relatively high levels (3-8 mg/L) of biologically active H GF into the culture medium. There combinant HGF induces pronounced mor phological changes and scattering of primary cultures of rat, mouse, a nd human hepatocytes within 24 h after plating and stimulates DNA synt hesis in these cells with the same magnitude as native HGF derived fro m human placenta or rabbit serum. The human recombinant HGF produced b y the insect cells is N-glycosylated, binds to heparin like native HGF , and is recognized by polyclonal antiserums raised against human or r abbit HGF as assessed by immunoblot, ELISA, and immunoneutralization e xperiments. Metabolic radiolabeling with L-[S-35]methionine (pulse-cha se experiments) as well as Western blot analysis indicates that the re combinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cul tured in serum-free medium. However, when the infected insect cells ar e cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterod imeric form. Addition of serum to the baculovirus-expressed single-cha in [I-125] HGF in a cell-free system results in conversion to the hete rodimeric two-chain form, and the activation is prevented by the serin e protease inhibitor PMSF. Incubation of I-125-labeled pro-HGF with ra t liver or spleen extracts resulted in conversion of pro-HGF to the he terodimeric two-chain form. A truncated form of HGF containing the N-t erminal portion of HGF (kringles 1-3) was also produced in the same ex pression system. This deleted HGF, by itself, did not have any detecta ble biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful produ ction of bioactive recombinant HGF in large quantities, which will all ow purification on the milligram scale of pro-HGF and will permit futu re studies to elucidate pathways involved in HGF activation by its tar get tissues.