title: Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli.
abstract: 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.
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Saiko
authors.initials: S
authors.lastname: Sugawara
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ito
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Sato
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Kano
authors.initials: K
authors.lastname: Kasuga
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Kojima
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan. Electronic address: makoba@akita-pu.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D016475:3T3 Cells; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D051519:Fibroblast Growth Factor 4 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006493:Heparin / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology; D014327:Trophoblasts / Q000166:cytology
chemical_list: C496446:Fgf4 protein, mouse; D051519:Fibroblast Growth Factor 4; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D006493:Heparin
keywords: Escherichia coli; Fibroblast growth factor 4; Mouse; Recombinant; Truncate