There is no disclosure anywhere in this document of mutations which might be made to affect the thermal stability. This document discloses measurement of the thermal stability of their preferred scaffold by differential scanning calorimetry (page 48, lines 31 to 32 and FIG. In particular, this document discloses three preferred synthetic proteins as SEQ ID No. WO 2014/125290 discloses scaffold proteins derived from plant cystatins. There is no teaching whatsoever regarding the variation or modulation of the thermal stability in this document. Thermostability in this document is solely disclosed as a property which can be measured in order to assess the scaffold protein's resistance to being deformed by the target peptide (see paragraph bridging pages 6 to 7 of WO 2006/131749). This document discloses a thermostability assay (page 32, first paragraph) and shows data on the heat stability of STM (page 35, lines 9 to 12). WO 2006/131749 discloses the use of Stefin A as a scaffold protein, and discloses several mutations useful for ensuring biological neutrality of the scaffold. Thermal stability is mentioned in only one paragraph in this document, merely as a one of several properties which might be measured in order to assess a scaffold protein's resistance to being deformed by a target peptide (WO 2009/136182 page 13, first paragraph). This document makes no teachings whatsoever regarding alteration of thermal stability of the scaffold protein. In particular, this document teaches the special advantages of a G4R mutant. In particular, the disclosures in this document focus on the “position 4 mutation”, which corresponds to the G4 site of Stefin A or the W4 site of STM. WO 2009/136182 discloses modified Stefin A scaffold proteins. These pairs of simultaneous mutations have been disclosed as exhibiting high expression in E. Simultaneous mutation of E78A and L80R has been disclosed for scaffold SQT in WO 2009/136182, as well as disclosing simultaneous mutation of L82R and T83S in the SQM scaffold (paragraph bridging pages 22 to 23 of WO 2009/136182). WO 2009/136182 as well as WO 2006/131749 have each previously disclosed mutation of the V48 site of wild-type Stefin A, in particular to V48D which is useful for the abolition of domain-swap dimerisation (WO 2006/131749 page 35, line 25). WO 2009/136182 discloses the mutation of prior art scaffold STM using D48L and G50S, which are disclosed as leading to increased expression in the bacterial system (page 22, lines 12 to 13). The invention also relates to various methods and nucleic acids.

Scaffold protein definition