title: Generation of aminoterminally truncated, stable types of bioactive bovine and porcine fibroblast growth factor 4 in Escherichia coli.
abstract: Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro(32) to Leu(206) ) proteins without a secretory signal peptide at the aminoterminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser(54) and Leu(55) in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu(55) to Leu(206) ) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs.
authors.affiliation: Graduate School of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
authors.forename: Saiko
authors.initials: S
authors.lastname: Sugawara
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ito
authors.forename: Shiori
authors.initials: S
authors.lastname: Sato
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sato
authors.forename: Akira
authors.initials: A
authors.lastname: Sasaki
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Fukuda
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Miki
authors.initials: M
authors.lastname: Sakatani
authors.forename: Masashi
authors.initials: M
authors.lastname: Takahashi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D051519:Fibroblast Growth Factor 4 / Q000096:biosynthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013552:Swine
chemical_list: D000596:Amino Acids; D051519:Fibroblast Growth Factor 4; D011994:Recombinant Proteins
keywords: Escherichia coli; FGF4; bovine; porcine; recombinant; stable; truncate