FUNCTIONAL AND BIOPHYSICAL CHARACTERIZATION OF RECOMBINANT HUMAN HEPATOCYTE GROWTH-FACTOR ISOFORMS PRODUCED IN ESCHERICHIA-COLI

Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic c ells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal(Nf domain through kringle 1 (NK1) or through kringle 2 (NK2), We report the high-level expression in Escherichia call of NK1 and NK2, as well as the individu al kringle 1 (K1) and N domains of HGF. All proteins accumulated as in soluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gelfiltration steps. Characterization of the purified proteins indicated chemical and physi cal homogeneity, and analysis by CD suggested native conformations, Al though the K1 and N-terminal domains of HGF have limited biological ac tivity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologicall y active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated b y mitogenicity, cell scatter, and receptor binding and activation assa ys. These data indicate that all four bacterially produced HGF derivat ives are well suited for detailed structural analysis.