US 11,788,081 B2
Protein macrocyclization
Tom Norbert Grossmann, Amstelveen (NL); Marta Pelay Gimeno, Leiden (NL); Sven Hennig, Amsterdam (NL); and Saskia Antonie Neubacher, Amstelveen (NL)
Assigned to Stichting VU, Amsterdam (NL)
Appl. No. 17/48,284
Filed by Stichting VU, Amsterdam (NL)
PCT Filed Apr. 18, 2019, PCT No. PCT/NL2019/050229
§ 371(c)(1), (2) Date Oct. 16, 2020,
PCT Pub. No. WO2019/203645, PCT Pub. Date Oct. 24, 2019.
Claims priority of application No. 18168298 (EP), filed on Apr. 19, 2018.
Prior Publication US 2021/0102185 A1, Apr. 8, 2021
Int. Cl. C12N 9/96 (2006.01); C12N 9/52 (2006.01); C12N 15/113 (2010.01)
CPC C12N 9/52 (2013.01) [C12N 15/113 (2013.01); C12Y 304/2207 (2013.01)] 5 Claims
 
1. A method for increasing the stability of a native or modified polypeptide or combination of polypeptides against denaturation, wherein the polypeptide or combination of polypeptides exhibits a natural tertiary structure having at least two secondary structures, said method comprising:
a) identifying within the polypeptide or combination of polypeptides in its natural tertiary structure three cysteine residues being located in the at least two distinct secondary structures having a spacial proximity for reaction with a trivalent thiol-reactive cross-linker having a C3 symmetric core, and
b) contacting said polypeptide or combination of polypeptides with the trivalent thiol-reactive cross-linker such that the linker forms covalent bonds with each of the three cysteine residues in a manner to stabilize the natural tertiary structure of the polypeptide or combination of polypeptides against denaturation compared to the stability of the polypeptide or combination of polypeptides without the reacted cross-linker, wherein the alpha-C atoms of the three cysteine residues form a triangle with side lengths between 6 to 23 Angstroms.