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2 results on '"Genenase I"'

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

Author

Hayata, Daichika, Fukuta, Kazuhiro, Matsumoto, Kunio, Adachi, Eri, Hanada, Keigo, Adachi, Kiichi, and Nakamura, Toshikazu

Subjects
*LIVER cells, *GROWTH factors, *PROTEOLYTIC enzymes, *HYDROLASES
Abstract

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 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. [Copyright &y& Elsevier]

Published
2008
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2. Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

Author

Toshikazu Nakamura, Kazuhiro Fukuta, Kunio Matsumoto, Eri Adachi, Kiichi Adachi, Keigo Hanada, and Daichika Hayata

Subjects
Serum, medicine.medical_treatment, Blotting, Western, Molecular Sequence Data, Activation, Bioengineering, Spodoptera, Cleavage (embryo), Protein Engineering, Applied Microbiology and Biotechnology, Models, Biological, law.invention, Cell Line, law, medicine, Animals, Humans, Amino Acid Sequence, Protein Precursors, Genenase I, Recombinant Hepatocyte Growth Factor, Serine protease, chemistry.chemical_classification, Hepatocyte growth factor, biology, Growth factor, Serine Endopeptidases, Subtilisin, General Medicine, Recombinant Proteins, Amino acid, medicine.anatomical_structure, Biochemistry, chemistry, Hepatocyte, biology.protein, Recombinant DNA, Electrophoresis, Polyacrylamide Gel, Biotechnology, medicine.drug
Abstract

金沢大学がん研究所附属分子標的がん医療研究開発センター, 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. © 2007 Elsevier B.V. All rights reserved.

Published
2008

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