Highly stable polypeptide scaffolds

2:

• The polypeptide scaffold of claim 1 further comprising E90.

3:

• The polypeptide scaffold of claim 1 in which the amino acid residues of Group A protect a hydrophobic core via mediation of interactions with water.

4:

• The polypeptide scaffold of claim 1 in which the amino acid residues of Group B provide favourable interactions amongst the N and C terminal strands of the molecule at elevated temperatures.

5:

• The polypeptide scaffold of claim 1 in which the amino acid residues of Group C provide a complementary salt bridge network on the surface of β-sheet 2 which restricts unfolding of the molecule at high temperatures.

7:

• The polypeptide scaffold of claim 6 comprising the amino acid sequence of residues 14-98 of SEQ ID NO:
• 2 or a variant thereof, with non-essential residues shown in lower case.
• X1-13psppgn LrVtdvtsts vtlswepppg pitgYRvEYR eaggewkEvt vpgsetsYtv tglkpgteYE fRvRavngag EgppssVsVtt (SEQ ID NO:2).

8:

• A polypeptide comprising the polypeptide scaffold of claim 1 incorporating one or more insertions or substitutions in one or more loop regions of the scaffold.

9:

• An isolated nucleic acid molecule encoding the polypeptide scaffold of claim 1 or complementary to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 or capable of hybridising to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 under selected stringency conditions.

10:

• A vector comprising the nucleic acid molecule of claim 9.

11:

• A host cell comprising and optionally transformed with the nucleic acid molecule of the claim 9.

12:

• A method of making the stable polypeptide scaffold of claim 1, the method comprising culturing a host cell to produce the polypeptide scaffold or polypeptide and recovering the scaffold or polypeptide, wherein the host cell comprises and optionally is transformed with a nucleic acid molecule encoding the polypeptide scaffold of claim 1 or complementary to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 or capable of hybridising to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 under selected stringency conditions.

13:

• A polypeptide scaffold or polypeptide produced by the method of claim 12.

14:

• A method of generating a library of the polypeptide scaffolds of claim 1, the method comprising incorporating randomised codons in order to produce polypeptide variants, comprising the steps of introducing randomised codons into a nucleic acid molecule at selected positions, wherein the nucleic acid molecule encodes the polypeptide scaffold of claim 1 or is complementary to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 or is capable of hybridising to the nucleic acid molecule encoding the polypeptide scaffold of claim 1 under selected stringency conditions, and propagating copies of the nucleic acid molecule to form a library of nucleic acid molecules encoding variant scaffold proteins.

15:

• A scaffold library comprising the polypeptide scaffold of claim 1.

16:

• A method of generating a scaffold molecule that binds to a particular target by panning the scaffold library of claim 15 with the target and detecting binders.

17:

• An isolated nucleic acid molecule encoding the polypeptide of claim 8 or complementary to the nucleic acid molecule encoding the polypeptide of claim 8 or capable of hybridising to the nucleic acid molecule encoding the polypeptide of claim 8 under selected stringency conditions.

18:

• A scaffold library generated by the method of claim 14.

19:

• A method of generating a scaffold molecule that binds to a particular target by panning the scaffold library of claim 18 with the target and detecting binders.

20:

• A host cell comprising and optionally transformed with the vector of claim 10.