Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

• Published in: Journal of biotechnology 2008-02, Vol.133 (4), p.478-485
• Main Authors: Hayata, Daichika, Fukuta, Kazuhiro, Matsumoto, Kunio, Adachi, Eri, Hanada, Keigo, Adachi, Kiichi, Nakamura, Toshikazu
• Format: Article
• Language: English
• Subjects: Activation; Amino Acid Sequence; Animals; Biological and medical sciences; Biotechnology; Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Genenase I; Hepatocyte growth factor; Hepatocyte Growth Factor - genetics; Hepatocyte Growth Factor - metabolism; Humans; Models, Biological; Molecular Sequence Data; Protein Engineering - methods; Protein Precursors - metabolism; Recombinant Proteins - metabolism; Serine Endopeptidases - metabolism; Serum; Spodoptera; Subtilisin - metabolism
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.11.006

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 α- and β-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 α-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.