Recombinant FGF2-G3 (145 aa) protein

Summary

• High purity thermostable FGF2-G3 protein comprising 145 aa form of FGF-2 (Uniprot: P09038) with nine stabilizing amino acid substitutions
• 16 kDa
• >98%, by SDS-PAGE quantitative densitometry
• Expressed in E. coli
• Animal origin-free (AOF) and carrier protein-free
• Manufactured in Qkine's Cambridge, UK laboratories
• Lyophilized from Tris, NaCl, CyS, mannitol
• Resuspend in sterile-filtered water at >50 µg/ml, add carrier protein if desired, prepare single use aliquots and store frozen at -20 °C (short-term) or -80 °C (long-term).

Handling and Storage FAQ

Featured applications

• Induced pluripotent stem cell culture and maintenance

WT FGF2 (Qk025) and FGF2-G3 (Qk052) were diluted in conditioned media and incubated at 37^oC for 2 or 7 days before being serially diluted and then assayed in triplicate for activity using the Promega serum response element luciferase reporter assay in transfected HEK293T cells. Firefly luciferase activity was measured and normalized to the control Renilla luciferase activity.

Protein background

FGF-2 (also known as basic FGF or bFGF) is an essential growth factor for maintainin human embryonic stem cell (hESC) and induced pluripotency stem cell (iPSC) pluripotency in feeder-free and chemically defined stem cell media. It is a core component of widely adopted media including mTESR (Ludwig et al. 2006), StemPRO (Wang et al. 2007) and E8 (Chen et al. 2011). However, FGF-2 is inherently unstable and prone to proteolytic degradation and aggregation. This fundamental biochemical instability, and therefore low half-life in culture media (<10 h), is an important contribution to the need for frequent media changes and challenges in improving homogeneity during stem cell proliferation and subsequent differentiation.

To improve the stability of FGF-2 for stem cell media and regenerative medicine applications, Dvorak and colleagues at Masaryk University used computer-assisted protein engineering to identify an optimal set of nine amino acid substitutions that stabilize FGF-2. These substitutions were designed to avoid structural changes to the FGF receptor 1 (FGFR1) and FGF receptor 2 (FGFR2) binding sites. This thermostable FGF-2 is known as FGF2-G3, or FGF2-STAB® (Dvorak et al. 2017). The biological activity of wild-type FGF-2 is <50% after 10h incubation with conditioned media. In contrast, no reduction in FGF2-G3 biological activity is observed after >7 days incubation with conditioned media at 37oC. Both FGF2-G3 and wild-type FGF-2 maintain hESC pluripotency and expression of pluripotency markers Oct-4 and nanog with equivalent efficacy (Dvorak et al. 2017).

In 2020, Paul Burridge and colleagues at Northwestern University, Chicago, published a protocol for B8 media. This iPSC maintenance media uses thermostable FGF2-G3, along with optimization of media component concentration and composition to reduce media cost and facilitate weekend-free stem cell culture regimes. (Kuo et al. 2020 and updated in Lyra-Leite et al. 2020).

To manufacture and provide FGF2-G3 for stem cell culture, including use in B8 media and emerging applications such as cultured meat, Qkine has licensed the patented FGF2-G3 technology from Enantis/Masaryk University. We have combined the excellent science behind the FGF2-G3 technology with our protein manufacture expertise to provide gold standard protein for use in cell culture media. We have removed His tags present in academic forms of the protein, as these may give rise to issues for scientists translating discoveries to the clinical or scale-up. His tags also introduce unnecessary scientific uncertainty.

Background references

1. Ludwig, T. E. et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185–187 (2006). doi.org/10.1038/nbt1177

2. Ludwig, T. E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006). doi.org/10.1038/nmeth902

3. Wang L, Schulz TC, Sherrer ES et al. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood. 2007 Dec 1;110(12):4111-9. doi: 10.1182/blood-2007-03-082586.

4. Chen G, Gulbranson DR, Hou Z et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011 May;8(5):424-9. doi: 10.1038/nmeth.1593.

5. Dvorak P, Bednar D, Vanacek P, et al. Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnology and Bioengineering. 2018; 115: 850–862. https://doi.org/10.1002/bit.26531

6. Kuo H H, Gao X, DeKeyser J-M, K. Fetterman A, et al. Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture, Stem Cell Reports 2020 https://doi.org/10.1016/j.stemcr.2019.12.007

7. Lyra-Leite D M, Fonoudi H, Gharib M, Burridge P W. An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells, STAR Protocols 2021. https://doi.org/10.1016/j.xpro.2020.100213

Publications using Recombinant FGF2-G3 (145 aa) protein (Qk052)

• Refined home-brew media for cost-effective, weekend-free hiPSC culture and genetic engineering
  Truszkowski L, Bottini S, Bianchi S et al. 
  DOI: https://doi.org/10.12688/openreseurope.18245.1

FAQ

What is FGF-2?
Fibroblast growth factor 2 (FGF-2), also known as basic fibroblast growth factor (bFGF) is a growth factor and signaling protein.

Where is FGF-2 found?
FGF-2 is expressed in a developmental and tissue specific manner. It’s expression is tightly controlled in normal tissues and it can be detected in all major tissues.

What does FGF-2 do?
FGF-2 is essential for normal embryonic development. It has roles in cell survival and proliferation, angiogenesis, tumorigenesis, wound healing and tissue repair.

What does FGF-2 bind to?
FGF-2 binds to and signals though all four of the FGF receptors FGFR1-4.

What is the function of the FGF receptor?
FGFRs phosphorylate specific tyrosine residues and activate the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways.

How is FGF-2 used in stem cell culture?
FGF-2 is used to maintain the pluripotency of stem cells in culture.