title: Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I.
abstract: Hepatocyte growth factor (HGF) is biosynthesized as a biologically inactive, single-chain form (pro-HGF). Its activation is associated with cleavage at Arg494-Val495 into a two-chain mature form composed of disulfide-linked alpha- and beta-chains. Because serum is a major source of HGF activator (the predominant serine protease responsible for the processing of pro-HGF), serum-free production of recombinant, two-chain HGF had not been established. In this study, to enable serum-free production of two-chain HGF, we generated engineered human pro-HGFs that can be specifically cleaved and activated by Genenase I. Since Genenase I specifically cleaves the C-terminus of the His-Tyr sequence, which does not exist in human HGF, Arg494 (the C-terminus of the HGF alpha-chain) was replaced by His-Tyr, Ala-Ala-His-Tyr, Pro-Gly-His-Tyr, or Pro-Gly-Ala-Ala-His-Tyr. Genenase I cleaved engineered pro-HGFs specifically at the replaced amino acid sequences, forming a disulfide-linked two-chain form. The cleavage was most efficient in the case of the Pro-Gly-Ala-Ala-His-Tyr sequence, and cleaved HGFs displayed biological activities identical to those of wild-type HGF. Considering a potential medical application of HGF, the present technique is valuable because it enables the production of recombinant, two-chain HGF entirely without serum and extends the choice of host cells and organisms for recombinant production.
authors.affiliation: Saito Laboratory, Research & Development, Kringle Pharma, Inc., 7-7-15 Saitoasagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Daichika
authors.initials: D
authors.lastname: Hayata
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Fukuta
authors.forename: Kunio
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Eri
authors.initials: E
authors.lastname: Adachi
authors.forename: Keigo
authors.initials: K
authors.lastname: Hanada
authors.forename: Kiichi
authors.initials: K
authors.lastname: Adachi
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D018411:Spodoptera; D020860:Subtilisin / Q000378:metabolism*
chemical_list: C066855:HGF protein, human; D011498:Protein Precursors; D011994:Recombinant Proteins; C086013:pro-hepatocyte growth factor; D017228:Hepatocyte Growth Factor; D012697:Serine Endopeptidases; D020860:Subtilisin