Common Amino Acid Sequences Deduced from Coding Exons of the Porcine<i>FGF4</i>Gene in Two Breeds and Production of the Encoded Protein in<i>Escherichia coli</i>

Biotech and App Biochem

et al. 2014

Self Cite

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.

show abstract

Section: Construction Of Bacterial Expression Vectors For Bovine and mentioning

confidence: 99%

Section: Protein Analysesmentioning

confidence: 99%

Section: Cell Growth Assaymentioning

confidence: 99%

“…AB633206) and pigs (AB745732) [14,15]. The amino acid sequences of mature types of bovine and porcine FGF4 proteins comprised of 206 amino acid residues display 96% homology.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Biotech and App Biochem

et al. 2014

Self Cite

show abstract

Identification of Site-Specific Degradation in Bacterially Expressed Human Fibroblast Growth Factor 4 and Generation of an Aminoterminally Truncated, Stable Form

Appl Biochem Biotechnol

et al. 2013

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for tumorigenesis in humans and the development of mammalian embryos. The secreted, mature form of human FGF4 is thought to be comprised of 175 amino acid residues (proline(32) to leucine(206), Pro(32)-Leu(206)). Here, we found that bacterially expressed, 6× histidine (His)-tagged human FGF4 (Pro(32)-Leu(206)) protein, referred to as HishFGF4, was unstable such as in phosphate-buffered saline. In these conditions, site-specific cleavage, including between Ser(54) and Leu(55), in HishFGF4 was identified. In order to generate stable human FGF4 derivatives, a 6× His-tagged human FGF4 (Leu(55)-Leu(206)), termed HishFGF4L, was expressed in Escherichia coli. HishFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HishFGF4L was considered as sufficiently stable. HishFGF4L exerted significant mitogenic activities in mouse embryonic fibroblast Balb/c 3T3 cells. In the presence of PD173074, an FGF receptor inhibitor, the growth-stimulating activity of HishFGF4L disappeared. Taken together, we suggest that HishFGF4L is capable of promoting cell growth via an authentic FGF signaling pathway. Our study provides a simple method for the production of a bioactive human FGF4 derivative in E. coli.

show abstract

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

Journal of Bioscience and Bioengineering

et al. 2014