Thermostable FGF2 polypeptide, use thereof
Inventors
Dvorak, Petr • Krejci, Pavel • Balek, Lukas • Eiselleova, Livia • Konecna, Zaneta • DVORAK, PAVEL • Bednar, David • BREZOVSKY, Jan • Sebestova, Eva • CHALOUPKOVA, Radka • STEPANKOVA, Veronika • Vanacek, Pavel • PROKOP, Zbynek • DAMBORSKY, Jiri • Bosakova, Michaela
Assignees
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Publication Number
US-11746135-B2
Publication Date
2023-09-05
Expiration Date
Abstract
The invention provides an isolated thermostable polypeptide possessing FGF2 activity and having at least 85% sequence identity to SEQ ID NO: 2 (FGF2 wt) or a functional fragment thereof, and comprising at least one amino acid substitution R31L and the use thereof in the cell biology research, regenerative medicine and related medical applications or cosmetics. Further it discloses a culture medium comprising subjected FGF2 suitable for culturing a human pluripotent stem cells involving both human embryonic stem cells and induced pluripotent stem cells.
Core Innovation
The invention relates to engineered thermostable FGF2 polypeptides that possess FGF2 activity. The thermostable polypeptides have at least a specified sequence identity to SEQ ID NO: 2 and comprise required amino acid substitutions, including R31L and optionally additional substitutions such as H59F, to increase thermal stability as reflected in melting temperature (Tm) measurements.
The invention further provides thermostable polypeptides with defined substitution patterns, including single-point and multi-point mutants. Specific examples include variants designated as FGF2 CS1/CS2 and higher-point variants such as FGF2 CS3/CS4/CS5, where the substitutions are selected from a defined set including residues such as V52T, H59F, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, and T121P, characterized for thermal stability using DSC/TSA and related thermal shift approaches.
The invention also discloses thermo-stabilized FGF2 variants that preserve FGF2 activity under elevated temperature conditions and are used in a culture medium for culturing human pluripotent stem cells. In this context, the polypeptide supports maintenance of an undifferentiated state, including preservation of pluripotency markers such as Oct4 and Nanog and improved proliferation results, with reduced need for daily medium changes or repeated supplementation as described.
Claims Coverage
The provided independent claims cover thermostable FGF2-active polypeptides defined by SEQ ID NO: 2 sequence identity and the presence of R31L, with further constraints based on the number and selection of additional specified substitutions. They also include culture-medium formulations for culturing human pluripotent stem cells in an undifferentiated state using the claimed thermostable polypeptides at an effective amount.
Thermostable FGF2 polypeptide with R31L and high sequence identity
A thermostable polypeptide possessing FGF2 activity and having at least 92% sequence identity to SEQ ID NO: 2, and comprising at least an amino acid substitution R31L.
Thermostable FGF2 polypeptide with R31L plus at least eight further specified substitutions
A thermostable polypeptide possessing FGF2 activity, having SEQ ID NO: 2, and comprising at least an amino acid substitution R31L, and comprising at least eight further amino acid substitutions selected from a group consisting of V52T, H59F, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, T121P.
Thermostable FGF2 polypeptide with R31L plus at least ten further specified substitutions
A thermostable polypeptide possessing FGF2 activity, having SEQ ID NO: 2, and comprising at least an amino acid substitution R31L, and comprising at least ten further amino acid substitutions selected from a group consisting of V52T, H59F, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, T121P.
Culture medium containing the thermostable FGF2 polypeptide for undifferentiated human pluripotent stem cells
A culture medium for culturing human pluripotent stem cells while keeping them undifferentiated contains an effective amount of a thermostable polypeptide (as defined in claims 1, 7, or 10) at a concentration of 1.0 ng/ml to 100 ng/ml.
Across the independent claims, coverage is centered on thermostable FGF2-active polypeptides defined by SEQ ID NO: 2 and the presence of R31L, with further constrained substitution sets based on a minimum number of additional specified amino acid substitutions. The claims also extend to a culture medium containing such thermostable polypeptides at an effective concentration to support undifferentiated culturing of human pluripotent stem cells.
Stated Advantages
Increased melting temperature (Tm) as a measure of thermal stability.
Preserved FGF2 activity under elevated temperature conditions.
Maintenance of undifferentiated morphology and pluripotency markers (Oct4 and Nanog) in human PSC culture.
Improved proliferation with thermo-stabilized FGF2 variants described (e.g., CS2/CS4).
Reduced need for daily medium changes or repeated supplementation in the described culture context.
Documented Applications
Culturing human pluripotent stem cells (hESC and iPSC) in an undifferentiated state using a culture medium containing an effective amount of a thermostable FGF2 polypeptide.
Using thermo-stabilized FGF2 polypeptides in culture conditions such that pluripotency markers (Oct4 and Nanog) are maintained and undifferentiated morphology is supported, with improved proliferation as described.
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