Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli

Sugawara, Saiko; Iwamoto, Toshihiko; Sato, Suguru; Sato, Yuki; Kasuga, Kano; Kojima, Ikuo; Kobayashi, Masayuki

doi:10.1016/j.jbiosc.2013.10.009

Cited by 8 publications

(19 citation statements)

References 29 publications

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“…In fact, aminoterminally truncated human FGF4 (Leu 55 to Leu 206 ) and mouse FGF4 (Leu 51 to Leu 202 ) proteins, which contain the core domains of other members of the FGF family, exhibited a growth-stimulatory effect on mouse embryonic fibroblast Balb/c 3T3 cells [18,19]. Therefore, we tried to produce aminoterminally truncated bovine and porcine FGF4 (Leu 55 to Leu 206 ) derivatives in E. coli in order to create stable recombinant FGF4 proteins and to investigate their biological activities.…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid analysis of FGF4 derivatives and peptide sequences for the aminotermini of proteins were performed as reported previously [18,19].…”

Section: Protein Analysesmentioning

confidence: 99%

“…The absorbance was measured using a microplate reader at 450 and 655 nm as a background. Mitogenic activity of the FGF4 derivatives and the effect of PD173074 on cell growth, evaluated using the WST-1 assay, correlated well with changes in the numbers of fibroblast cells [24]. The statistical significance of the difference among sample means was determined using one-way analysis of variance followed by the Tukey-Kramer multiple comparison test.…”

Section: Production Of Fgf 4 Derivatives In E Coli (A) Production Omentioning

confidence: 99%

“…FGF4, including those in humans, mice, cattle, and pigs, contains a single N-linked glycosylation signal in the aminoterminal region of the mature protein, although glycosylation is not essential to its biological activities [17]. Therefore, aminoterminally truncated human and mouse FGF4 proteins, which lost the potential N-linked glycosylation site, expressed in Escherichia coli still had mitogenic activity [18,19].…”

Section: Introductionmentioning

confidence: 99%

“…An application of these FGF4 derivatives for the generation of bovine and porcine TS cells is expected. On the other hand, our recent preliminary observations suggested that the spontaneous degradation of both FGF4 derivatives occurred in Dulbecco's phosphate-buffered saline (PBS; Ca 2+ and Mg 2+ free, 0.14 M NaCl); HisbFGF4 and HispFGF4 were degraded at least to a protein with an apparent molecular weight (Mr) of 17 kDa, as well as shown in bacterially expressed mature types of human and mouse FGF4 proteins [18,19]. In the current study, in order to generate stable recombinant bovine and porcine FGF4 proteins and to investigate their biological activities, cleavage sites in the aminoterminal …”

Section: Introductionmentioning

confidence: 99%

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Generation of aminoterminally truncated, stable types of bioactive bovine and porcine fibroblast growth factor 4 in Escherichia coli

Sugawara

Iwamoto

Sato

et al. 2014

Biotech and App Biochem

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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.

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Section: Resultsmentioning

confidence: 99%

“…Amino acid analysis of FGF4 derivatives and peptide sequences for the aminotermini of proteins were performed as reported previously [18,19].…”

Section: Protein Analysesmentioning

confidence: 99%

Section: Production Of Fgf 4 Derivatives In E Coli (A) Production Omentioning

confidence: 99%