Computer designed stabilized proteins and method for producing same

Claim 2

A computer based method for evaluating a protein to determine whether said protein contains at least two target amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein stabilizing disulfide bond, said bond containing two cysteine residues; said method comprising the steps of: (a) ascertaining relative three-dimensional positions of atoms of a pair of cysteine residues of a candidate disulfide bond; (b) (i) examining a candidate pair of amino acid residues of said protein to determine whether they contain atoms whose relative three-dimensional positions possess a geometric conformation which is similar to a geometric conformation possessed by atoms of said cysteine residues of said candidate disulfide bond (a); (ii) examining whether replacement of the candidate pair of amino acid residues of said protein by said cysteine residues of said candidate disulfide bond can be accommodated, to thereby permit the formation of a potentially stabilizing disulfide bond, without creating a conformation of said protein molecule having unacceptable steric hindrance; (c) repeating said examinations (b)(i) and (b)(ii) for each candidate pair of amino acid residues of said protein, and for each ascertained candidate disulfide bond (a); (d) ranking each candidate pair of amino acid residues of said protein for its capacity to be replaced with a potentially stabilizing candidate disulfide bond, without undesirably altering said tertiary conformation of the protein molecule; and (e) using said ranking (d) to identify a target pair of amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein-stabilizing disulfide bond.

Claim 3

The method of claim 1 which additionally comprises: (f) identifying at least one target amino acid residue of said protein which could been placed by a cysteine residue thereby permitting the formation of a potentially protein-stabilizing disulfide bond; and (g) producing a protein molecule wherein said identified target amino acid residue has been replaced with a cysteine residue, said replacement permitting the formation of said potentially protein-stabilizing disulfide bone.

Claim 4

The method of claim 2 which additionally comprises: (h) forming the disulfide bond.

Claim 5

A computer based method for producing an engineered protein having a potentially protein-stabilizing disulfide bond which comprises: (a) using a computer based method to evaluate structure of said protein to determine whether said protein contains at least two target amino acid residues replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially stabilizing disulfide bond; said method comprising the steps: (1) examining each selected pair of amino acids in said protein to determine if they contain atoms whose relative three-dimensional positions possess a geometric conformation similar to a geometric conformation possessed by atoms of a disulfide bond. (2) examining any pair of amino acids found to contain said certain atoms identified in step (1) to determine whether said disulfide bond can be accommodated without creating unacceptable steric hindrance, (3) permitting an expert operator (i) to view any possible disulfide bond which can be accommodated without altering a tertiary conformation of said protein, and (ii) to rank said viewed possible disulfide bond from most likely to stabilize an engineered protein to least likely to stabilize said protein, and (4) evaluating said ranked possible disulfide bond according to expert rule criterion; and (b) producing an engineered protein molecule wherein at least one of said target amino acid residues has been replaced by a cysteine residue, said replacement permitting the formation of a potentially protein-stabilizing disulfide bond.

Claim 6

A computer based method for producing an engineered protein having a potentially protein-stabilizing disulfide bond which comprises: (a) using a computer based method to evaluate structure of said protein to determine whether said protein contains at least two target amino acid residues replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially stabilizing disulfide bond; said method comprising the steps: (1) examining each selected pair of amino acids in said protein to determine if they contain atoms whose relative three-dimensional positions possess a geometric conformation similar to a geometric conformation possessed by atoms of a disulfide bond. (2) examining any pair of amino acids found to contain said certain atoms identified in step (1) to determine whether said disulfide bond can be accommodated without creating unacceptable steric hindrance, (3) permitting an expert operator (i) to view any possible disulfide bond which can be accommodated without altering a tertiary conformation of said protein, and (ii) to rank said viewed possible disulfide bond from most likely to stabilize an engineered protein to least likely to stabilize said protein, and (4) evaluating said ranked possible disulfide bond according to expert rule criterion; and (b) producing an engineered protein molecule wherein at least one of said target amino acid residues has been replaced by a cysteine residue, said replacement permitting the formation of a potentially protein-stabilizing disulfide bond.

Claim 7

The method of claim 4 wherein said expert rule criteria of step (4) comprises the steps: (a) evaluating a possible disulfide linkage to determine whether formation of said linkage would require loss of an evolutionally conserved amino acid residue; or (b) evaluating a possible disulfide linkage to determine whether formation of said linkage would result in loss of a favorable hydrophobic interaction.

Claim 8

A protein of increased stability, wherein said protein is produced through the use of a computer based method for evaluating said protein to determine whether said protein contains at least two target amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein-stabilizing disulfide bond, said bond containing two cysteine residues; said method comprising the steps of: (a) ascertaining relative three-dimensional positions of atoms of a pair of cysteine residues of a candidate disulfide bond; (b) (i) examining a candidate pair of amino acid residues of said protein to determine whether they contain atoms whose relative three-dimensional positions possess a geometric conformation which is similar to a geometric conformation possessed by atoms of said cysteine residues of said candidate disulfide bond (a); (ii) examining whether replacement of the candidate pair of amino acid residues of said protein by said cysteine residues of said candidate disulfide bond can be accommodated, to thereby permit the formation of a potentially stabilizing disulfide bond, without undesirably altering a tertiary conformation of said protein; (c) repeating said examinings (b)(i) and (b)(ii) for each candidate pair of amino acid residues of said protein, and for each ascertained candidate disulfide bond (a); (d) ranking each candidate pair of amino acid residues of said protein for its capacity to be replaced with a potentially stabilizing candidate disulfide bond, without undesirably altering the tertiary conformation of the protein molecule; (e) using said ranking (d) to identify a target pair of amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein-stabilizing disulfide bond; (f) identifying at least one target amino acid residue of said protein which could be replaced by a cysteine residue thereby permitting the formation of a potentially protein-stabilizing disulfide bond; (g) producing a protein molecule wherein said identified target amino acid residue has been replaced with a cysteine residue, said replacement permitting the formation of said potentially protein-stabilizing disulfide bond, and (h) forming the disulfide bond, to thereby form said protein of increased stability.

Claim 9

A protein of increased stability, wherein said protein is produced through the use of a computer based method for evaluating said protein to determine whether said protein contains at least two target amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein-stabilizing disulfide bond, said bond containing two cysteine residues; said method comprising the steps of: (a) ascertaining relative three-dimensional positions of atoms of a pair of cysteine residues of a candidate disulfide bond; (b) (i) examining a candidate pair of amino acid residues of said protein to determine whether they contain atoms whose relative three-dimensional positions possess a geometric conformation which is similar to a geometric conformation possessed by atoms of said cysteine residues of said candidate disulfide bond (a); (ii) examining whether replacement of the candidate pair of amino acid residues of said protein by said cysteine residues of said candidate disulfide bond can be accommodated, to thereby permit the formation of a potentially stabilizing disulfide bond, without undesirably altering a tertiary conformation of said protein; (c) repeating said examinings (b)(i) and (b)(ii) for each candidate pair of amino acid residues of said protein, and for each ascertained candidate disulfide bond (a); (d) ranking each candidate pair of amino acid residues of said protein for its capacity to be replaced with a potentially stabilizing candidate disulfide bond, without undesirably altering the tertiary conformation of the protein molecule; (e) using said ranking (d) to identify a target pair of amino acid residues, replacement of at least one of which with a cysteine residue would be sufficient to permit formation of at least one potentially protein-stabilizing disulfide bond; (f) identifying at least one target amino acid residue of said protein which could be replaced by a cysteine residue thereby permitting the formation of a potentially protein-stabilizing disulfide bond; (g) producing a protein molecule wherein said identified target amino acid residue has been replaced with a cysteine residue, said replacement permitting the formation of said potentially protein-stabilizing disulfide bond, and (h) forming the disulfide bond, to thereby form said protein of increased stability.

Claim 10

A nucleic acid sequence which encodes the protein of claim 6.

Claim 11

The nucleic acid of claim 7 wherein said nucleic acid is DNA.

Claim 12

The nucleic acid of claim 7 wherein said nucleic acid RNA.

Claim 13

The protein of claim 6 wherein said protein is selected from the group consisting of an enzyme and a binding protein.

Claim 14

The protein of claim 10 wherein said protein is a binding protein.

Claim 15

The binding protein of claim 11 wherein said binding protein is a DNA binding protein.

Claim 16

A nucleic acid sequence which encodes the binding protein of claim 12.

Claim 17

The binding protein of claim 11 wherein said binding protein is selected from the group consisting of receptor binding proteins, hormone binding proteins, antigen binding proteins and hapten binding proteins.

Claim 18

A nucleic acid sequence which encodes the binding protein of claim 14.

Claim 19

A nucleic acid sequence which encodes the binding protein of claim 11.

Claim 20

The protein of claim 10 wherein said protein is an enzyme.

Claim 21

A nucleic acid sequence which encodes the enzyme of claim 17.

Claim 22

The enzyme of claim 17 wherein said enzyme is a protease.

Claim 23

The protease of claim 19 wherein said protease is a serine protease homologous to subtilisin.

Claim 24

The protease of claim 20 wherein said protease contains at least one disulfide bond at a position determined by: (a) obtaining the amino acid sequence of said protease; (b) aligning the amino acid sequence of said protease with the amino acid sequence of subtilisin to maximize amino acid homology; and (c) determining the position of said disulfide bond by identifying amino acid positions of said protein which have a geometry similar to at least one disulfide bond selected from the group consisting of: (i) a disulfide bond between residues 22 and 87 of said subtilisin; or (ii) a disulfide bond between residues 206 and 216 of said subtilisin.

Claim 25

A nucleic acid sequence which encodes the protease of claim 19.

Claim 26

The protease of claim 19 wherein said protease is subtilisin.

Claim 27

The subtilisin of claim 23 wherein said subtilisin is selected from the group consisting of subtilisin 7159, subtilisin 8307, subtilisin 7181, and subtilisin 8310.

Claim 28

The subtilisin of claim 23 wherein said subtilisin contains at least one disulfide bond selected from the group consisting of: (a) a disulfide bond between residues 22 and 87; and (b) a disulfide bond between residues 206 and 216.

Claim 30

A nucleic acid sequence which encodes the subtilisin of claim 23.

Claim 31

A nucleic acid sequence which encodes the subtilisin of claim 24.