Protein surface remodeling
First Claim
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1. A method of improving the stability of a protein of interest, the method comprising steps of:
- (1) identifying non-conserved surface residues of a protein of interest by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position; and
(2) replacing a plurality of non-conserved, surface residues identified in step (1) with a natural amino acid residue that is positively charged at physiological pH, whereby the method creates a modified protein of interest having a theoretical net charge of at least about +10 at physiological pH as compared to the theoretical net charge of the original protein of interest.
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Abstract
Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.
76 Citations
35 Claims
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1. A method of improving the stability of a protein of interest, the method comprising steps of:
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(1) identifying non-conserved surface residues of a protein of interest by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position; and (2) replacing a plurality of non-conserved, surface residues identified in step (1) with a natural amino acid residue that is positively charged at physiological pH, whereby the method creates a modified protein of interest having a theoretical net charge of at least about +10 at physiological pH as compared to the theoretical net charge of the original protein of interest. - View Dependent Claims (30, 31, 32, 33)
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2. A method of improving the stability of a protein of interest, the method comprising steps of:
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(1) identifying non-conserved surface residues of a protein of interest by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position; and (2) replacing a plurality of non-conserved, surface residues identified in step (1) with a natural amino acid residue that is negatively charged at physiological pH, whereby the method creates a modified protein of interest having a theoretical net charge of less than about −
10 at physiological pH as compared to the theoretical net charge of the original protein of interest.
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3. A method of improving the stability of a protein of interest, the method comprising steps of:
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(1) identifying non-conserved surface residues of the protein of interest by comparing the amino acid sequence of the protein with at least one other amino acid sequence of the protein from the same protein family or a different species, wherein a residue is non-conserved if less than or equal to 50% of the amino acid sequences have the same amino acid sequence in a particular position; (2) assigning a hydrophobicity value to each of the identified non-conserved surface residues; and (3) replacing at least one surface residue with a natural amino acid residue that is charged at physiological pH, wherein the residues being replaced are all mutated into the same type of residue, wherein the type of residue is either positively charged residues or negatively charged residues; and (4) creating a modified protein of interest having a theoretical net charge of at least about +10 or less than about −
10 at physiological pH as compared to the theoretical net charge of the original protein of interest. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 34, 35)
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Specification
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Current AssigneePresident and Fellows of Harvard College (Harvard Corporation)
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Original AssigneePresident and Fellows of Harvard College (Harvard Corporation)
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InventorsLiu, David R., Phillips, Kevin John, Lawrence, Michael S.
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Primary Examiner(s)Riggs, II, Larry D
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Application NumberUS12/303,047Publication NumberTime in Patent Office3,049 DaysField of SearchUS Class Current1/1CPC Class CodesC07K 14/43595 from coelenteratae, e.g. me...C12N 9/93 Ligases (6)C12Y 603/02003 Glutathione synthase (6.3.2.3)
