Recombinant FGF2-G3 (154aa) protein

Summary

• High purity thermostable recombinant FGF2-G3 protein comprising 154 aa form of FGF-2 (Uniprot: P09038) with nine stabilizing amino acid substitutions
• 17 kDa
• >98%, by SDS-PAGE quantitative densitometry
• Animal origin-free (AOF) and carrier protein-free
• Expressed in E. coli
• Manufactured in our 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).

Featured applications

• Expansion of induced pluripotent, embryonic and mesenchymal stem cells

WT FGF-2 (Qk027) and FGF2-G3 (Qk053) were diluted in conditioned media and incubated at 37oC for 2 or 7 days before FGF-2 activity was assayed in triplicate using the Promega serum response element luciferase reporter assay in transfected HEK293T cells. Firefly luciferase activity was normalized to the control Renilla luciferase activity.

Protein background

FGF-2 (also known as basic FGF or bFGF) is an essential growth factor for maintaining 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 [1, 2], StemPRO [3] and E8 [4]. 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® [5]. 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 [5].

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 [6, 7].

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 (154 aa) protein (Qk053)

• A chemically defined and xeno-free hydrogel system for regenerative medicine
  Ong J, Gibbons G, Siang LY et al.
  DOI: https://doi.org/10.1101/2024.05.28.596179
• Cytogenetic resource enables mechanistic resolution of changing trends in human pluripotent stem cell aberrations linked to feeder-free culture
  Stavish D, Price CJ, Gelezauskaite G et al.
  DOI: https://doi.org/10.1101/2023.09.21.558777
• Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing
  van Bree N, Oppelt AS, Lindström S et al.
  DOI: https://doi.org/10.1093/neuonc/noae210
• Ephrin-A2 and Phosphoantigen-Mediated Selective Killing of Medulloblastoma by γδT Cells Preserves Neuronal and Stem Cell Integrity
  Boutin L, Liu M, Merville JD et al.
  DOI: https://doi.org/10.1101/2024.10.12.617193
• Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q
  Stavish D, Price CJ, Gelezauskaite G, Alsehli H et al.
  DOI: https://doi.org/10.1016/j.stemcr.2024.06.003
• Modeling the selective growth advantage of genetically variant human pluripotent stem cells to identify opportunities for manufacturing process control
  Beltran-Rendon C, Price CJ, Glen K et al.
  DOI:  https://doi.org/10.1016/j.jcyt.2024.01.010
• mTOR activity paces human blastocyst stage developmental progression
  Iyer DP, Khoei HH, van der Weijden VA et al.
  DOI: https://doi.org/10.1016/j.cell.2024.08.048
• 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
• Vortioxetine: A Potential Drug for Repurposing for Glioblastoma Treatment via a Microsphere Local Delivery System
  Wang Y, Siebzehnrubl D, Weller M et al.
  DOI: https://doi.org/10.1021/acsbiomaterials.5c00068
• Parkinson’s associated protein DJ-1 regulates intercellular communication via extracellular vesicles in oxidative stress
  Page T, Musi CA, Bakker SE et al.
  DOI: https://doi.org/10.21203/rs.3.rs-5669239/v1

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.