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US 2016/0097042 A1Published application
Internal protein tags
AssigneePromega Corporation
InventorsAndrew S. Dixon, Lance P. Encell, Thomas Machleidt, Marie Schwinn, Keith Wood, Monika Wood, Kris Zimmerman
Life of this patent
Estimating the term…
Abstract
Provided herein are substantially non-luminescent peptide/polypeptide tags that are inserted internally within a protein of interest or between N-terminal and C-terminal peptides/polypeptides. Interaction of the internally-inserted tag with a complement polypeptide/peptide that is also substantially non-luminescent results in the formation a bioluminescent reporter complex).
Claims40
A polypeptide comprising:
- (a) an N-terminal segment, a C-terminal segment, and an internal tag, wherein the internal tag comprises an amino acid sequence having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2 inserted within a protein of interest;
- wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide of SEQ ID NO:
- 440;
- or
- (b) an N-terminal segment, a C-terminal segment, and an internal tag, wherein the internal tag comprises an amino acid sequence having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440 inserted within a protein of interest wherein a detectable bioluminescent signal is produced in the presence of a substrate when the detection peptide contacts a polypeptide of SEQ ID NO:
- 2.
5 more elements
(canceled)
(canceled)
(canceled)
20-37. (canceled)
The method of claim 77, for detecting a stable interaction between a first amino acid sequence and a second amino acid sequence comprising:
- (a) creating an internal fusion by inserting an internal tag into the first amino acid sequence, such that said internal tag is neither at the N-terminus not the C-terminus of the first amino acid sequence, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide of SEQ ID NO:
- 440;
- (b) creating a second fusion of the second amino acid sequence and a complement polypeptide, wherein the complement polypeptide has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the complement polypeptide contacts a peptide of SEQ ID NO:
- 2;
- (c) placing the internal fusion, second fusion, and a coelenterazine substrate in conditions that allow for a possible stable interaction to occur between the first amino acid sequence and the second amino acid sequence;
- and
- (d) detecting, if present, a bioluminescent signal emitted, wherein detection of the bioluminescent signal indicates a stable interaction between the first amino acid sequence and the second amino acid sequence.
6 more elements
-43. (canceled)
The method of claim 77, for detecting a stable interaction between a first amino acid sequence and a second amino acid sequence comprising:
- (a) creating an internal fusion by inserting an internal tag into the first amino acid sequence, such that said internal tag is neither at the N-terminus not the C-terminus of the first amino acid sequence, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a peptide of SEQ ID NO:
- 2;
- (b) creating a second fusion of the second amino acid sequence and a complement peptide, wherein the complement peptide has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the complement peptide contacts a polypeptide of SEQ ID NO:
- 440;
- (c) placing the internal fusion, second fusion, and a coelenterazine substrate in conditions that allow for a possible stable interaction to occur between the first amino acid sequence and the second amino acid sequence to;
- and
- (d) detecting, if present, a bioluminescent signal emitted, wherein detection of the bioluminescent signal indicates a stable interaction between the first amino acid sequence and the second amino acid sequence.
6 more elements
-49. (canceled)
The method of claim 77, for detecting a target polypeptide in a sample comprising:
- (a) creating an internal fusion by inserting an internal tag into the target polypeptide, such that said internal tag is neither at the N-terminus not the C-terminus of the target polypeptide, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a peptide of SEQ ID NO:
- 2;
- (b) adding to said sample:
- (i) a complement peptide that has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, and
- (ii) a coelenterazine substrate;
- (c) detecting, if present, a bioluminescent signal emitted, wherein detection of the bioluminescent signal indicates the presence of the target polypeptide in the sample.
5 more elements
-53. (canceled)
The method of claim 77, for detecting a target polypeptide in a sample comprising:
- (a) creating an internal fusion by inserting an internal tag into the target polypeptide, such that said internal tag is neither at the N-terminus not the C-terminus of the target polypeptide, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a peptide of SEQ ID NO:
- 440;
- (b) adding to said sample:
- (i) a complement polypeptide that has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, and
- (ii) a coelenterazine substrate;
- (c) detecting, if present, a bioluminescent signal emitted, wherein detection of the bioluminescent signal indicates the presence of the target polypeptide in the sample.
5 more elements
-57. (canceled)
A detection reagent comprising:
- (a) (i) a complement polypeptide comprising an amino acid sequence having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, wherein a detectable bioluminescent signal is produced, in the presence of a substrate, when the polypeptide contacts a peptide of SEQ ID NO:
- 2, and (ii) a substrate for a bioluminescent complex produced by said polypeptide and a peptide of SEQ ID NO:
- 2;
- or (b) (i) a complement peptide comprising an amino acid sequence having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, wherein a detectable bioluminescent signal is produced, in the presence of a substrate, when the peptide contacts a polypeptide of SEQ ID NO:
- 440, and (ii) a substrate for a bioluminescent complex produced by said peptide and a polypeptide of SEQ ID NO:
- 440.
5 more elements
(canceled)
The method of claim 77, for detecting alteration of an interaction between a first amino acid sequence and a second amino acid sequence by a potential inhibitory agent comprising:
- (a) creating an internal fusion by inserting an internal tag into the first amino acid sequence, such that said internal tag is neither at the N-terminus not the C-terminus of the first amino acid sequence, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide of SEQ ID NO:
- 440;
- (b) creating a second fusion of the second amino acid sequence and a complement polypeptide, wherein the complement polypeptide has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, and wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the complement polypeptide contacts a peptide of SEQ ID NO:
- 2;
- (c) placing the internal fusion, second fusion, and a coelenterazine substrate in conditions that allow for a possible stable interaction to occur between the first amino acid sequence and the second amino acid sequence;
- (d) detecting, if present, a bioluminescent signal emitted;
- wherein detection of the bioluminescent signal indicates a stable interaction between the first amino acid sequence and the second amino acid sequence;
- (e) adding the potential inhibitory agent to the internal fusion, second fusion, and a coelenterazine substrate;
- (f) detecting, if present, a bioluminescent signal emitted;
- and
- (g) comparing the bioluminescent signals of steps (d) and (f), wherein decrease in bioluminescent signal from step (d) to step (f) indicates inhibition of the interaction between the first amino acid sequence and the second amino acid sequence by the potential inhibitory agent.
10 more elements
-62. (canceled)
The method of claim 77, for determining the structural conformation of a first amino acid sequence comprising:
- (a) creating an internal fusion by inserting an internal tag into the first amino acid sequence, such that said internal tag is neither at the N-terminus not the C-terminus of the first amino acid sequence, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2, wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide of SEQ ID NO:
- 440, wherein a first structural conformation of the first amino acid sequence prevents access to the internal tag, and wherein a second structural conformation of the first amino acid sequence allows access to the internal tag;
- (b) placing the internal fusion and either (i) a complement polypeptide having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440 or (ii) a second fusion of a second amino acid sequence and the complement polypeptide in the presence of a coelenterazine substrate;
- (c) detecting, if present, a bioluminescent signal emitted, wherein absence of the bioluminescent signal indicates the first amino acid sequence is adopting the first structural conformation, and wherein presence of the bioluminescent signal indicates the first amino acid sequence is adopting the second structural conformation.
3 more elements
(canceled)
The method of claim 77, for determining the structural conformation of a first amino acid sequence comprising:
- (a) creating an internal fusion by inserting an internal tag into the first amino acid sequence, such that said internal tag is neither at the N-terminus not the C-terminus of the first amino acid sequence, wherein the internal tag has less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 440, wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a peptide of SEQ ID NO:
- 2, wherein a first structural conformation of the first amino acid sequence prevents access to the internal tag, and wherein a second structural conformation of the first amino acid sequence allows access to the internal tag;
- (b) placing the internal fusion and either (i) a complement peptide having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2 or (ii) a second fusion of a second amino acid sequence and the complement peptide in the presence of a coelenterazine substrate;
- (c) detecting, if present, a bioluminescent signal emitted, wherein absence of the bioluminescent signal indicates the first amino acid sequence is adopting the first structural conformation, and wherein presence of the bioluminescent signal indicates the first amino acid sequence is adopting the second structural conformation.
3 more elements
-68. (canceled)
A polypeptide comprising an N-terminal segment, a C-terminal segment, and two or more internal tags, wherein the internal tags comprise amino acid sequences having less than 100% and greater than 30% sequence identity with SEQ ID NO:
- 2 inserted within a protein of interest;
- wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when one or more of the internal tags contact a polypeptide of SEQ ID NO:
- 440.
-76. (canceled)
Dependencies are inferred from the claim text.