RRC ID 10962
著者 Hayata D, Fukuta K, Matsumoto K, Adachi E, Hanada K, Adachi K, Nakamura T.
タイトル Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I.
ジャーナル J Biotechnol
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.
巻・号 133(4)
ページ 478-85
公開日 2008-2-29
DOI 10.1016/j.jbiotec.2007.11.006
PII S0168-1656(07)01745-2
PMID 18178280
MeSH Amino Acid Sequence Animals Blotting, Western Cell Line Electrophoresis, Polyacrylamide Gel 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* Spodoptera Subtilisin / metabolism*
IF 3.503
引用数 2
WOS 分野 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
リソース情報
ヒト・動物細胞 Mv.1.Lu(NBL-7)(RCB0996)