Identification of site-specific degradation in bacterially expressed human fibroblast growth factor 4 and generation of an aminoterminally truncated, stable form.
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.
Graduate School of Bioresource Sciences, Akita Prefectural University, Akita, 010-0195, Japan.
Saiko
S
Sugawara
Toshihiko
T
Ito
Shiori
S
Sato
Yuki
Y
Sato
Kano
K
Kasuga
Ikuo
I
Kojima
Masayuki
M
Kobayashi
D000818:Animals; D041702:BALB 3T3 Cells; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D051519:Fibroblast Growth Factor 4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D015202:Protein Engineering; D055550:Protein Stability; D013379:Substrate Specificity
C496445:FGF4 protein, human; D051519:Fibroblast Growth Factor 4; D010446:Peptide Fragments