Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro^<31>-Leu^<202>) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser^<50> and Leu^<51> was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu^<51>_Leu^<202>),termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.

• 公益社団法人日本生物工学会の論文

著者

• Sato Yuki

  Graduate School Of Biological Sciences Nara Institute Of Science And Technology

• Kasuga Kano

  Graduate School of Bioresource Sciences, Akita Prefectural University

• Sugawara Saiko

  Graduate School of Bioresource Sciences, Akita Prefectural University

• Kojima Ikuo

  Graduate School of Bioresource Sciences, Akita Prefectural University

• Kobayashi Masayuki

  Graduate School of Bioresource Sciences, Akita Prefectural University

• ITO Toshihiko

  Graduate School of Bioresource Sciences, Akita Prefectural University

• Sato Shiori

  Graduate School of Bioresource Sciences, Akita Prefectural University

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