Computer designed stabilized proteins and method for producing same
First Claim
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1. 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.
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Abstract
Proteins, such as enzymes having enhanced stability are designed through the use of a computer method. The method identifies amino acid residues of a protein which may be replaced with a cysteine residue in order to promote the formation of a protein-stabilizing disulfide bond. The computer-designed, stabilized proteins can be produced using recombinant DNA technology.
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Citations
28 Claims
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1. 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. - View Dependent Claims (2, 3)
- said method comprising the steps of;
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4. A computer based method for producing an engineered protein having a potentially protein-stabilizing disulfide bond which comprises:
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(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. - View Dependent Claims (5)
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6. 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. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
- said method comprising the steps of;
Specification