Means and methods for generating improved proteins

Patent metadata

Field	Value
Publication number	US-9957495-B2
Application number	US-201314395049-A
Country	US
Kind code	B2
Filing date	Apr 18, 2013
Priority date	Apr 18, 2012
Publication date	May 1, 2018
Grant date	May 1, 2018

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.

First claim

The invention claimed is:

1. 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.

2. 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.

Assignees

• Vib Vzw
• Univ Leuven Kath
• Univ Leuven Kath

Inventors

• Rousseau Frederic
• Schymkowitz Joost
• Ganesan Ashok
• Siekierska Aleksandra
• De Smet Frederik
• Van Durme Joost

Classifications

• G06F19/16

  Physics · mapped topic

• C07K14/43595

  from coelenteratae, e.g. medusae · CPC title

• C40B30/02

  Chemistry & Metallurgy · mapped topic

• C12N9/2465 Primary

  acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22) · CPC title

• C07K14/32

  from Bacillus (G) · CPC title