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US 10,294,266 B2Granted
Engineering surface epitopes to improve protein crystallization
AssigneeThe Trustees of Columbia University in the City of New York
InventorsVictor Naumov, William Nicholson Price, Samuel K. Handelman, John Frances Hunt
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Abstract
The invention provides for methods and systems for engineering target proteins, based on protein sequence characteristics that influence the likelihood of obtaining a crystal suitable for X-ray structure solution, to improve protein crystallization, as well as related material.
Claims7
A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties, comprising:
- a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
- b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
- i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
- ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
- iii. using a computer program to predict the secondary structure of the target protein based on its sequence;
- and
- iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein;
- and
- c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces;
- and
- d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence;
- and
- e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system.
10 more elements
A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties comprising:
- a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
- b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
- i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
- ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
- iii. using a computer program to predict the secondary structure of the target protein based on its sequence;
- and
- iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein;
- and
- c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces;
- and
- d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence;
- and
- e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system, and the expressed protein is crystallized and its structure is determined with high-resolution X-ray crystallography.
10 more elements
A method for increasing the number of high-quality crystal-packing motifs in a target protein to improve its crystallization properties, comprising
- a. providing a sub-epitope library containing local crystal-packing motifs in the PDB that span at most two-successive regular secondary structural elements and flanking loops, wherein each sub-epitope is ranked by p-value according to its overrepresentation in crystal-packing interfaces formed by crystal structures in the PDB that do not have excessively close inter-protein contacts;
- b. identifying one or more specific candidate sites in the sequence of the target protein for introduction of each sub-epitope in the library by:
- i. using a computer program to search a protein sequence database for proteins homologous to the target protein;
- ii. using a computer program to perform a multiple sequence alignment of the target sequence with the homologous proteins identified by the search program;
- iii. using a computer program to predict the secondary structure of the target protein based on its sequence;
- and
- iv. specifying exact sites in the target protein for introduction of a sub-epitope from the library based on the occurrence of residues similar to those in the sub-epitope at aligned positions in one of the homologous protein sequences and on conservation of the secondary structure of the sub-epitope in the target protein;
- and
- c. prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on the overrepresentation p-value of the sub-epitope in crystal-packing interfaces;
- and
- d. further prioritizing sub-epitopes for introduction via mutagenesis at the specific sites identified for that sub-epitope in the target protein based on whether the number of sub-epitopes of equal or better overrepresentation p-value is increased by the required mutations in the target sequence;
- and
- e. obtaining a mutant protein sequence of the target protein based on the sub-epitope prioritization steps and wherein the mutant protein sequence is expressed in an expression system to provide a mutant protein with the mutant protein sequence and is crystallized and its structure is determined with high-resolution X-ray crystallography.
10 more elements
Dependencies are inferred from the claim text.