Means and methods for generating improved proteins
AssigneesVib Vzw, Katholieke Universiteit Leuven, KU LEUVEN R&D
InventorsFrederic Rousseau, Joost Schymkowitz, Ashok Ganesan, Aleksandra Siekierska, Frederik De Smet, Joost Van Durme
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Abstract
The disclosure provides a general method for the production of protein variants with a reduced aggregation propensity without affecting the thermodynamic stability of the variant with respect to the wild-type protein.
Claims2
A method of producing a reduced aggregating variant of a wild-type protein, which wild-type protein has a high-resolution crystallographic structure available, the wild-type protein comprising at least two beta-aggregation regions, the method comprising:
- a) determining the at least two beta-aggregation regions in the wild-type protein;
- b) performing systematic mutation screens of aggregation gatekeeper residues R, K, E, D and P of all amino acids belonging to the determined beta-aggregation regions to generate a list of variant proteins thereof, wherein each variant protein thereof has at least one amino acid position in the at least two beta-aggregation regions changed to either R, K, E, D, or P;
- c) calculating, for each of the variant proteins, a predicted aggregation score and a predicted change in thermodynamic stability with respect to the wild-type protein;
- and
- d) producing, based upon the generated list, a reduced aggregating variant having, at the same time, a maximally reduced predicted aggregation and a maximal preservation of thermodynamic stability, so as to eliminate mutations from the list that thermodynamically destabilize the native structure with the use of an atomic force field.
A method of producing a reduced-aggregating variant of a wild-type protein, the wild-type protein having two or more beta-aggregation-regions and further having a high-resolution crystallographic structure available, the method comprising:
- conducting a systematic mutation screen of aggregation gatekeeper residues R, K, E, D and P of all amino acids belonging to a beta-aggregation region determined to be in the wild-type protein to identify variant proteins of the wild-type protein, wherein each variant protein identified has at least one amino acid position in the beta-aggregation region substituted with either R, K, E, D, or P;
- calculating, for each of the identified variant proteins, a predicted aggregation score and a predicted change in thermodynamic stability in comparison to the wild-type protein;
- and
- synthesizing a variant protein thus calculated to have both a maximally reduced predicted aggregation score and a maximal preservation of thermodynamic stability in comparison to the wild-type protein, so as to eliminate variant proteins that thermodynamically destabilize the wild-type protein's native structure with the use of an atomic force field.
Dependencies are inferred from the claim text.