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

Abstract

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.

Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

“…Briefly, hESCs were differentiated into endoderm using 100 ng/ml activin A for 3 days and then further differentiated into CDX2 ϩ intestinal tissue using 2 M Chir99021 (04-0004-10; Stemgent) plus 500 ng/ml fibroblast growth factor 4 (FGF4) for 4 to 6 days. FGF4, used to differentiate the intestinal tissue, was either obtained from R&D Systems (235-F4) or purified in the laboratory as previously described (12). During intestinal specification, three-dimensional spheroids emerged in the culture dish.…”

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

“…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.…”

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

“…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].…”

“…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 …”