Stable proteins and methods for designing same

1. A method of producing a non-naturally occurring designed protein having an improved stability compared to the stability of the corresponding wild-type protein, wherein the designed protein comprises at least one modified polypeptide chain having at least six amino acid substitutions relative to an original polypeptide chain in the wild-type protein, the method comprising:

A: providing a protein expression vector suitable for use in a protein expression system, said vector comprises protein sequence data obtained by computationally designing said modified polypeptide chain starting from said original polypeptide chain, wherein said computationally designing comprises the steps of:

(i) determining unsubstitutable positions and substitutable positions in an amino acid sequence of said original polypeptide chain;

(ii) determining at least one position-specific amino acid alternative for each of said substitutable positions, and determining a position-specific stability scoring for each of said amino acid alternative;

(iii) combinatorially generating a plurality of designed sequences, each of said designed sequences corresponds to a modified polypeptide chain and comprises at least six amino acid substitutions each being one of said at least one position-specific amino acid alternative, and threading each of said designed sequences on a template structure of said original polypeptide chain, to thereby generate a plurality of designed structures;

(iv) sorting said plurality of designed structures according to a minimized energy scoring, said minimized energy scoring is determined by subjecting each of said designed structures to an energy minimization; and

(v) selecting at least one of said plurality of designed structures, corresponding to said modified polypeptide chain, based on said minimized energy scoring,

thereby obtaining an amino acid sequence of said modified polypeptide chain for use as sequence data input for providing said protein expression vector;

B: expressing the designed protein in said protein expression system using said protein expression vector, thereby producing the designed protein in said system, wherein said substitutions improved the stability of the designed protein relative to the corresponding wild-type protein, as determined by at least one of:

a thermal denaturation temperature of the designed protein being equal or higher than a thermal denaturation temperature of the wild type protein;

a solubility of the designed protein being equal or higher than a solubility of the wild type protein;

a degree of misfolding of the designed protein being equal or lower than a degree of misfolding of the wild type protein;

a half-life of the designed protein being equal or longer than a half-life of the wild type protein;

a specific activity of the designed protein being equal or higher than a specific activity of the wild type protein; and

a recombinant expression level of the designed protein being equal or higher than a recombinant expression level of the wild type protein.

2. The method of claim 1 , wherein said original polypeptide chain comprises at least 100 amino acids.

3. The method of claim 1 , wherein a selected modified polypeptide chain corresponds to designed structure having a minimal value for said minimized energy scoring.

4. The method of claim 3 , wherein said energy minimization is a global energy minimization.

5. The method of claim 1 , wherein said plurality of designed sequences is combinatorially generated under an acceptance threshold based on said stability scoring.

6. The method of claim 1 , wherein determining said unsubstitutable positions and said substitutable positions is based on a sequence alignment of a plurality of amino acid sequences homologous to the original polypeptide chain.

7. The method of claim 6 , wherein, for loop regions, said sequence alignment comprises amino acid sequences having sequence length equal to a corresponding loop in the original polypeptide chain.

8. The method of claim 6 , at least one of said unsubstitutable positions is determined based on said sequence alignment.

9. The method of claim 6 , wherein said sequence alignment is based on a non-redundant database of sequences.

10. The method of claim 9 , wherein said plurality of amino acid sequences comprises sequences having less than 30% sequence identity with respect to said original polypeptide chain.

11. The method of claim 10 , wherein said plurality of amino acid sequences is clustered using a threshold of 90-100%.

12. The method of claim 11 , wherein amino acid sequences having a coverage of less than 40% and a sequence identity of less than 15% are excluded from said plurality of amino acid sequences.

13. The method of claim 12 , wherein amino acid sequences having more than 5% gaps (INDELs) are excluded from said plurality of amino acid sequences.

14. The method of claim 1 , wherein at least one of said unsubstitutable positions is selected from the group consisting of a highly conserved position, an active-site position, a metal binding position, a ligand binding position, a substrates binding position, a DNA/RNA binding position, a structure stabilizing position and an antigenic determinant position.

15. The method of claim 1 , wherein determining said position-specific amino acid alternative is dictated by rules.

16. The method of claim 15 , wherein said rules comprise a position-specific scoring matrix (PSSM).

17. The method of claim 1 , wherein said position-specific stability scoring is determined based on an energy minimization.

18. The method of claim 17 , wherein said energy minimization is a local energy minimization.

19. The method of claim 18 , wherein said local energy minimization is effected for amino acid residues of the modified polypeptide chain having at least one atom being less than about 5 Å from at least one atom of said position-specific amino acid alternative.

20. The method of claim 1 , wherein said template structure is subjected to global energy minimization prior to said threading.

21. The method of claim 20 , wherein said template structure is an experimentally determined structure.

22. The method of claim 20 , wherein said template structure is computationally determined based on an experimentally determined structure of a naturally occurring homolog of the original polypeptide chain.

23. The method of claim 1 , wherein said energy minimization comprises at least one operation selected from the group consisting of bond length optimization, bond angle optimization, backbone dihedral angles optimization, amino acid side-chain packing optimization and rigid-body optimization of the modified polypeptide chain.

24. The method of claim 1 , wherein a shortest distance of Cα of at least one of said amino acid substitutions is at least 6 Å from a water-accessible surface of the designed protein.