Revision as of 12:52, 7 October 2021

Bovine growth factor FGF2 which induces cell proliferation

Profile

Name: FGF2 WT
Base Pairs: 828 bp
Origin: Escherichia coli, synthetic
Properties: Bovine growth factor which induces cell proliferation

Sequence and Features

Usage & Biology

Fibroblast growth factor 2 (FGF2) is one of 19 known members of the mammalian FGF family, involved in morphogenesis, development, angiogenesis and wound healing [1]. It is a mitogen and was first isolated in 1974, from the bovine pituitary and in 1988 human FGF2 was described for the first time [2]. There are four different FGF receptor tyrosine kinases (FGFR), to which FGFs bind and induce cell signaling. FGF2 binds FGFR1 and FGFR2 to induce proliferation [3]. FGFR2 consists of an extracellular domain, a transmembrane helix, and a catalytic intracellular tyrosine kinase domain [1]. The extracellular domain of the receptor consists of three immunoglobulin-like domains. FGF2 binds to domain 2 (D2) and domain 3 (D3), and the linker region connects the two domains [1] (figure 2).

Figure 2. Crystal structure of FGF2 (green) bound to FGFR2 (cyan). PDB: 1EV2.

When FGF2 binds to the extracellular part of the receptor it causes the receptor to dimerise with another FGF-bound FGFR2, leading to conformational changes which activates the receptor in its intracellular domain and cell signaling is initiated [4]. Through an intracellular signaling cascade gene regulation occurs in favour of cell proliferation, resulting in cell growth [4] (figure 3).

IMAGE - binding & signaling

Inducing cell growth through FGF2 signaling is utilized in the field of cellular agriculture, where the growth factor is used in the serum-free growth medium for cultivating meat. Similar to in a biological system, FGF2 induces cell growth when cultivating meat in a bioreactor. However, growth media is expensive, 55-95% of the production cost of cultivated meat comes from the growth medium [5]. To make serum-free media economically feasible on an industrial scale, the medium needs to be optimized. Being one of the most important and most expensive components, FGF2 is one of the targets for improvement [6].

To enable bovine FGF2 for research, a biobrick part to express bovine FGF2 has been designed and tested. This part will make FGF2 more accessible to support future research on medium optimization within cellular agriculture.

Experimental Results

Biobrick Assembly

The FGF2 biobrick part was designed inside a pUC plasmid together with the basic parts T7 promoter (BBa_J64997), Lac operon (BBa_K3630001) T7 RBS (BBa_K3257011), T7 T_phi terminator (BBa_B0016). To increase solubility of the protein during expression and purification, the FGF2 gene was fused with thioredoxin (BBa_K3934008), and to enable protein purification a 6xHis-tag (BBa_K3934015) was added. An enterokinase site (BBa_K3934016) was added to cleave off the 6xHis-tag and thioredoxin. The pUC plasmid also contains a gene for ampicillin resistance. The design was ordered from Integrated DNA Technologies (IDT).

The biological system used for biobrick assembly was E.coli DH5α competent cells. A T_phi terminator, an NdeI restriction site and an PstI restriction site were added to the 5’ end and the 3’ end of the FGF2 construct using PCR primers. The restriction sites and terminator were part of the primer overhang sequences. The PCR modified construct was treated with NdeI and PstI and ligated into a pET vector containing an IPTG inducible T7 promoter, a Lac operon, an RBS and a kanamycin resistance gene. The plasmid was re-ligated using DNA ligase and transformed into E.coli DH5α. To remove the risk of religation of the pET vector, the restriction enzyme treated pET was run through gel electrophoresis and the band corresponding to the part of the plasmid to use was extracted with gel purification. The properly assembled plasmid (figure 4) was verified using Sanger sequencing Mix2Seq Kit from Eurofins Genomics and then transformed into E. coli BL21 (DE3) pLysS competent cells from Promega which are optimized for protein expression. Transformation was also done using NEB BL21 (DE3) cells.

Figure 4. Assembled biobrick vector.