Methods for immobilizing polypeptides
First Claim
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1. A method for immobilizing a polypeptide to a surface, wherein the method comprises:
- contacting a polypeptide which comprises an ester or thioester, with an anchor molecule comprising a first nucleophilic group at a 2 or 3 position relative to a second nucleophilic group, wherein the ester or thioester undergoes a trans-esterification reaction with the first nucleophilic group, thus forming an intermediate compound in which the polypeptide is attached to the anchor molecule through the first nucleophilic group; and
attaching the anchor molecule to a surface.
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Abstract
This invention provides methods for immobilizing polypeptides, for forming arrays of polypeptides arranged on a support, and arrays produced using the methods of the invention. The immobilized polypeptides of the invention are generally in the same orientation, can be full-length and biologically active, and can be readily screened for a desired activity.
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Citations
85 Claims
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1. A method for immobilizing a polypeptide to a surface, wherein the method comprises:
- contacting a polypeptide which comprises an ester or thioester, with an anchor molecule comprising a first nucleophilic group at a 2 or 3 position relative to a second nucleophilic group,
wherein the ester or thioester undergoes a trans-esterification reaction with the first nucleophilic group, thus forming an intermediate compound in which the polypeptide is attached to the anchor molecule through the first nucleophilic group; and
attaching the anchor molecule to a surface. - View Dependent Claims (2, 3, 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, 30, 31, 32, 33, 34, 35, 80)
- contacting a polypeptide which comprises an ester or thioester, with an anchor molecule comprising a first nucleophilic group at a 2 or 3 position relative to a second nucleophilic group,
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36. An array of immobilized polypeptides attached to a surface, wherein the array comprises at least a first polypeptide species and a second polypeptide species and each of which polypeptide species are:
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attached to a separate region of the surface;
attached to the surface in the same orientation; and
are folded in a secondary structure as required for a biological activity. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45)
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- 46. An array of immobilized polypeptides attached to a surface which comprises a plurality of surface regions, wherein each surface region has attached thereto a polypeptide species and a polynucleotide that encodes the polypeptide species.
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50. A method for screening a library of nucleic acids to identify a nucleic acid that encodes a polypeptide having a desired activity, the method comprising:
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expressing a plurality of fusion proteins, each of which is encoded by an expression cassette that comprises;
a) a member of the library of nucleic acids;
b) an intein coding region; and
c) an open reading frame that encodes a polypeptide that is displayed on a surface of a replicable genetic package;
wherein the fusion proteins are displayed on the surface of a replicable genetic package; and
screening the replicable genetic packages to identify those that display a polypeptide having the desired activity. - View Dependent Claims (51, 52, 53, 54, 55)
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56. A nucleic acid that comprises an expression cassette, wherein the expression cassette comprises:
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an insertion site at which a polynucleotide can be introduced into the expression cassette;
an intein coding region, wherein the carboxyl terminus of the intein coding region is mutated so that it does not function as a splice junction for intein-mediated cleavage; and
an open reading frame that encodes a polypeptide that is displayed on a surface of a replicable genetic package;
wherein the introduction of a polynucleotide at the insertion site results in an open reading frame that encodes a fusion protein which comprises a polypeptide encoded by the polynucleotide, which polypeptide is attached at its carboxyl terminus to an amino terminus of the intein, and the surface-displayed polypeptide is attached to a carboxyl terminus of the intein. - View Dependent Claims (57, 58, 59)
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60. A method for immobilizing a polypeptide to a surface, wherein the method comprises:
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contacting a polypeptide which comprises an ester or thioester, with an anchor molecule comprising a first nucleophilic group at a 2 or 3 position relative to a second nucleophilic group, wherein the ester or thioester undergoes a trans-esterification reaction with the first nucleophilic group, thus forming an intermediate compound in which the polypeptide is attached to the anchor molecule through the first nucleophilic group;
wherein said intermediate compound undergoes an intramolecular rearrangement in which the second nucleophilic group on the anchor molecule displaces the first nucleophilic group, thus forming a bond between the anchor molecule and the polypeptide; and
attaching the anchor molecule to a surface.
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61. A method for immobilizing a polypeptide to a surface, wherein the method comprises:
contacting a polypeptide which comprises an ester or thioester, with an anchor molecule comprising a reactive group selected from the group consisting of a NH2—
NH—
R group and an aminooxy groupwherein R represents an anchor molecule, wherein the ester or thioester reacts with the reactive group, thus forming a compound comprising a polypeptide attached to the anchor molecule through the reactive group. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)
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77. A kit for use in immobilizing one or more polypeptides containing an ester or thioester to a surface of a substrate comprising:
an anchor molecule reagent for adapting said ester or thioester containing polypeptide to said surface, wherein said anchor molecule comprises a first nucleophilic group at a 2 or 3 position relative to a second nucleophilic group, wherein the ester or thioester of said one or more polypeptides undergoes a trans-esterification reaction with the first nucleophilic group, thus forming an intermediate compound in which the polypeptides are attached to the anchor molecules through the first nucleophilic group, wherein said anchor molecule is adapted for attachment to said surface of said substrate. - View Dependent Claims (78, 79, 81, 82, 83, 84, 85)
Specification