U.S. patent application number 17/522489 was filed with the patent office on 2022-03-03 for reactive peptide labeling.
The applicant listed for this patent is Promega Corporation. Invention is credited to Melanie Dart, Rod Flemming, Becky Godat, Virginia Kincaid, Nidhi Nath, Hui Wang, Wenhui Zhou.
Application Number | 20220065786 17/522489 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220065786 |
Kind Code |
A1 |
Wang; Hui ; et al. |
March 3, 2022 |
REACTIVE PEPTIDE LABELING
Abstract
Provided herein are sulfo n-hydroxysuccimidyl ester (sulfo-SE)
linked peptides, methods of synthesis thereof, and methods of using
such peptides for labeling of biomolecules. In particular, peptides
comprising non-alkyl group such as serine, threonine, cysteine,
tyrosine, glutamic acid, and aspartic acid are stably modified
(e.g., without autoreactivity) with a sulfo-SE group and used to
label or otherwise modify biomolecules.
Inventors: |
Wang; Hui; (Madison, WI)
; Nath; Nidhi; (Madison, WI) ; Flemming; Rod;
(Madison, WI) ; Godat; Becky; (Madison, WI)
; Zhou; Wenhui; (Madison, WI) ; Kincaid;
Virginia; (Madison, WI) ; Dart; Melanie;
(Madison w, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Promega Corporation |
Madison |
WI |
US |
|
|
Appl. No.: |
17/522489 |
Filed: |
November 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16698143 |
Nov 27, 2019 |
11175228 |
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17522489 |
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62772448 |
Nov 28, 2018 |
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International
Class: |
G01N 21/64 20060101
G01N021/64; C07K 7/08 20060101 C07K007/08; G01N 33/542 20060101
G01N033/542; C07K 1/13 20060101 C07K001/13; C07K 14/00 20060101
C07K014/00; C07D 207/46 20060101 C07D207/46 |
Claims
1. A composition comprising a peptide linked to a sulfo
n-hydroxysuccimidyl ester (sulfo-SE) group, wherein the peptide
does not comprise a cysteine or lysine residue.
2. The composition of claim 1, wherein the sulfo-SE is linked to
the N-terminus of the peptide.
3. The composition of claim 1, wherein the sulfo-SE is linked to
the C-terminus of the peptide.
4. The composition of claim 1, wherein the sulfo-SE is linked to an
amino acid sidechain of the peptide.
5. The composition of claim 1, wherein the peptide comprises at
least one non-alkyl amino acid selected from serine, threonine,
tyrosine, glutamic acid, arginine, histidine, tryptophan and
aspartic acid.
6. The composition of claim 5, wherein the at least one reactive
non-alkyl amino acid is a or tyrosine.
7. The composition of claim 5, wherein the at least one reactive
nucleophilic amino acid is an arginine.
8. The composition of claim 1, wherein the sulfo-SE group is linked
to the peptide by a non-peptide linker group.
9. The composition of claim 8, wherein the linker group comprises
and alkyl or heteroalkyl chain.
10. The composition of claim 8, wherein the linker comprises one or
more sidechain substituents.
11. The composition of claim 1, wherein the peptide is 4-50 amino
acids in length.
12. The composition if claim 11, wherein the peptide is 8-20 amino
acids in length.
13. The composition of claim 1, wherein the sulfo-SE is attached to
the N-terminus of the peptide.
14. The composition of claim 13, wherein the sulfo-SE is attached
to the N-terminus of the peptide via a linker group.
15. The composition of claim 1, wherein the peptides comprise a
fluorophore or chromophore conjugate.
16. The composition of claim 1, wherein the peptide is a component
of a biomolecular complex.
17. The composition of claim 14, wherein the peptide is a component
of a biomolecular complex.
18. The composition of claim 17, wherein the peptide comprises 5 or
fewer substitutions relative to SEQ ID NO: 10 (SmBiT).
19. The composition of claim 17, wherein one or more lysine of SEQ
ID NO: 1 are replaced with arginine.
20. The composition of claim 19, wherein the peptide comprises
Pep691 (SEQ ID NO: 23).
21. The composition of claim 19, wherein the peptide comprises
SmBiT (SEQ ID NO: 10).
22. The composition of claim 18, wherein the peptide is conjugated
to a fluorophore.
23. The composition of 22, wherein the peptide comprises
fluorophore conjugated to an arginine.
24. The composition of 23, wherein the peptide comprises
fluorophore conjugated to SEQ ID NO: 23.
25. The composition of 23, wherein the peptide comprises
fluorophore conjugated to SEQ ID NO: 10.
26. A method of labeling a biomolecule with a peptide comprising
contacting the biomolecule with a composition of one of claims
1-25, under conditions such that the sulfo-SE group reacts with an
amine on the biomolecule.
27. The method of claim 24, wherein a peptide composition of claim
14 contacts the biomolecule under conditions such that the sulfo-SE
group reacts with amine on the biomolecule.
28. The method of claim 26 or 27, wherein the amine is a primary
amine.
29. The method of claim 14, wherein the biomolecule is selected
from the group consisting of an antigen, an antibody, an antibody
fragment, a nanobody, a darpin, a non-antibody protein, a receptor,
a ligand, a toxin, a cytokine, a nucleic acid, a nucleoprotein
complex, a peptide, an amino acid, a sugar, a drug, and
streptavidin.
30. A method of labeling a peptide with a sulfo-SE moiety
comprising contacting the peptide with a sulfo-NHS compound under
conditions such that the hydroxy of the sulfo-NHS compound reacts
with the terminal amine of the peptide, wherein the peptide does
not comprise a cysteine or lysine residue.
31. The method of claim 20, wherein the peptide comprises at least
one reactive nucleophilic amino acid.
32. A composition comprising a biomolecule labeled with a peptide
of one of claims 1-25.
33. A method comprising contacting the composition of claim 32 with
an analyte.
34. The method of claim 33, wherein the analyte is selected from
the group consisting of an antigen, an antibody, an antibody
fragment, a nanobody, a darpin, a non-antibody protein, a receptor,
a ligand, a toxin, a cytokine, a nucleic acid, a nucleoprotein
complex, a peptide, an amino acid, a sugar, a drug, and
streptavidin.
35. The method of claim 35, wherein the analyte is linked to a
complementary polypeptide capable of forming a bioluminescent
complex with the peptide on the biomolecule.
36. The method of claim 35, further comprising contacting the
bioluminescent complex with a substrate for the bioluminescent
complex and detecting luminescence.
37. A composition comprising an analyte labeled with a peptide of
one of claims 1-25.
38. A method comprising contacting the composition of claim 37 with
a biomolecule.
39. The method of claim 38, wherein the biomolecule is linked to a
complementary polypeptide capable of forming a bioluminescent
complex with the peptide on the analyte.
40. The method of claim 39, further comprising contacting the
bioluminescent complex with a substrate for the bioluminescent
complex and detecting luminescence, fluorescence, and/or BRET.
41. A composition comprising an analyte labeled with a first
peptide of one of claims 1-25 and a biomolecule labelled with a
second peptide of one of claims 1-25, wherein the first and second
peptides are capable of forming a bioluminescent complex in the
presence of a complementary polypeptide.
42. A method comprising contacting the analyte and biomolecule of
claim 41 with the complementary polypeptide and forming the
bioluminescent complex.
43. The method of claim 42, further comprising contacting the
bioluminescent complex with a substrate for the bioluminescent
complex and detecting luminescence.
44. The method of one of claims 26-31, 33-36, 38-40, and 42-43,
wherein one or more of the peptides is a fluorophore or
chromophore-conjugated peptide.
45. The method of claim 44, further comprising detecting
fluorescence/light and/or BRET from the bioluminescent complex to
the fluorophore or chromophore.
46. The method of claim 45, wherein the number of labeling per
biomolecule is calculated by the number of fluorophore or
chromophore molecules per biomolecule.
47. The method of claim 44, the fluorophore molecule is a FAM,
TAMRA, ROX, silo-rhodamine, BODIPY, TOM, Dyomics dye, or a
carbon-rhodamine, but not limited to those fluorophores.
48. A method comprising (a) forming a bioluminescent complex of a
SEQ ID NO: 1 (SmBiT) labeled-analyte biomolecule and the
LgBiT-labeled analyte biomolecule specific antibody; (b) contacting
the bioluminescent complex with the analyte; (c) contacting the
bioluminescent complex with a substrate for the bioluminescent
complex; and (d) detecting light output from the bioluminescent
complex.
49. A method comprising (a) contacting an analyte with a SEQ ID NO:
1 (SmBiT) labeled-analyte specific antibody and a LgBiT-labeled
analyte-specific antibody, and forming a bioluminescent complex;
(b) contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (c) detecting light output from the
bioluminescent complex.
50. A method comprising: (a) contacting an analyte with a SEQ ID
NO: 1 (SmBiT) labeled-analyte specific antibody, a SEQ ID NO: 11
(HiBiT)-labeled analyte-specific antibody, and a polypeptide
copable for forming a bioluminescent complex with HiBiT and SmBiT;
(b) contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (c) detecting light output from the
bioluminescent complex.
51. A method comprising contacting a biomolecule labeled with a
composition of one of claims 1-25 with an analyte.
52. The method of claim 51, wherein the analyte is selected from
the group consisting of an antigen, an antibody, a non-antibody
protein, a receptor, a ligand, a toxin, a cytokine, a nucleic acid,
a peptide, an amino acid, a sugar, a drug, a nucleoprotein complex,
biotin, and streptavidin.
53. The method of claim 51, wherein the analyte biomolecule is
labeled with SEQ ID NO: 1 (SmBiT).
54. The method of claim 51, wherein (a) forming a bioluminescent
complex from a SEQ ID NO: 1 (SmBiT) labeled-analyte biomolecule and
a LgBiT-labeled analyte biomolecule specific antibody; (b)
contacting the bioluminescent complex with the analyte; (c)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (d) detecting light output from the
bioluminescent complex.
55. The method of claim 51, wherein (a) forming a bioluminescent
complex from SEQ ID NO: 1 (SmBiT) labeled- and LgBiT-labeled
analyte specific antibodies; (b) contacting the bioluminescent
complex with the analyte; (c) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; and (d) detecting
light output from the bioluminescent complex.
56. The method of claim 51, wherein (a) contacting an analyte with
SEQ ID NO: 10 (SmBiT)-labeled antibodies or receptors and SEQ ID
NO: 11 (HiBiT)-labeled antibodies or receptors; (b) contacting the
analyte with LgBiT to form a bioluminescent complex; (c) contacting
the bioluminescent complex with a substrate for the bioluminescent
complex; and (d) detecting light output from the bioluminescent
complex.
57. The method of claim 51, wherein a) contacting SmBiT- or
HiBiT-labeled-analyte biomolecule with a HiBiT- or SmBiT-labeled
analyte biomolecule specific antibody; (b) contacting with LgBiT to
form a bioluminescent complex; (c) contacting with the analyte (d)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (e) detecting light output from the
bioluminescent complex.
58. The method of claim 57, wherein the peptide is a fluorophore or
chromophore-conjugated peptide.
59. The method of claim 58, wherein the number of labeling per
biomolecule is calculated by the number of fluorophore or
chromophore molecules per biomolecule.
60. The method of claim 58, the fluorophore molecule is a FAM,
TAMRA, ROX, silo-rhodamine, BODIPY, TOM, Dyomics dye, or a
carbon-rhodamine, but not limited to those fluorophores.
61. The method of claim 51, wherein (a) forming a bioluminescent
complex from a fluorophore-conjugated SEQ ID NO: 1 (SmBiT)
labeled-analyte biomolecule and a LgBiT-labeled analyte biomolecule
specific antibody; (b) contacting the bioluminescent complex with
the analyte; (c) contacting the bioluminescent complex with a
substrate for the bioluminescent complex; and (d) detecting light
output from the bioluminescent complex.
62. The method of claim 51, wherein (a) contacting an analyte with
both a fluorophore-conjugated SEQ ID NO: 1 (SmBiT) labeled- and
LgBiT-labeled analyte specific antibodies, forming a bioluminescent
complex (b) contacting the bioluminescent complex with a substrate
for the bioluminescent complex; and (c) detecting light output from
the bioluminescent complex.
63. The method of claim 51, wherein (a) one of SEQ ID NO: 10
(SmBiT) and SEQ ID NO: 11 (HiBiT)-peptides is the
fluorophore-conjugated peptide; (b) the analyte contacts SEQ ID NO:
10 (SmBiT) and SEQ ID NO: 11 (HiBiT)-labeled antibodies, or
receptors, or combination, wherein one of the peptides is a
fluorophore-conjugated peptide; (b) contacting with LgBiT to form a
bioluminescent complex; (c) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; and (d) detecting
light output from the bioluminescent complex.
64. A method wherein the analyte biomolecule or analyte specific
antibody is labeled with fluorophore-conjugated SEQ ID NO: 10
(SmBiT) or SEQ ID NO: 11 (HiBiT), wherein (a) SEQ ID NO: 10 (SmBiT)
or SEQ ID NO: 11 (HiBiT)-labeled-analyte biomolecule contacts the
SEQ ID NO: 11 (HiBiT) or SEQ ID NO: 10 (SmBiT)-labeled analyte
biomolecule specific antibody, wherein one of the peptides is the
fluorophore-conjugated peptide; (b) contacting with LgBiT to form a
bioluminescent complex; (c) contacting with the analyte (d)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (e) detecting light output from the
bioluminescent complex.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 16/698,143, filed Nov. 27, 2019, now allowed,
which claims priority to U.S. Provisional Patent Application Ser.
No. 62/772,448, filed Nov. 28, 2018, each of which is hereby
incorporated by reference in its entireties.
SEQUENCE LISTING
[0002] The text of the computer readable sequence listing filed
herewith, titled "36664-303_SEQUENCE_LISTING_ST25", created Nov. 9,
2021, having a file size of 16,155 bytes, is hereby incorporated by
reference in its entirety.
FIELD
[0003] Provided herein are sulfo n-hydroxysuccimidyl ester
(sulfo-SE) linked peptides, methods of synthesis thereof, and
methods of using such peptides for labeling of biomolecules. In
particular, peptides comprising non-alkyl group such as serine,
threonine, tyrosine, glutamic acid, and aspartic acid are stably
modified (e.g., without autoreactivity) with a sulfo-SE group and
used to label or otherwise modify biomolecules.
BACKGROUND
[0004] The primary amine of lysine amino acids is a widely
available reactive moiety on biomolecules and reacts readily with
various agents, for example, an n-hydroxy succinimidyl ester (SE)
group. A reaction between primary amines on biomolecules and SE
containing fluorescent dyes, biotin, drugs, etc., have been used to
label proteins, antibodies, etc., for use in a variety of
applications. SE groups react with nucleophilic amino acid side
chains, such as arginine, lysine, histidine, cysteine, serine,
tyrosine, aspartic acid, glutamic acid, etc., therefore making SE
containing peptides for subsequent peptide labeling of biomolecules
has been avoided.
SUMMARY
[0005] Provided herein are sulfo n-hydroxysuccimidyl ester
(sulfo-SE) linked peptides, methods of synthesis thereof, and
methods of using such peptides for labeling of biomolecules. In
particular, peptides comprising non-alkyl group such as serine,
threonine, tyrosine, glutamic acid, and aspartic acid are stably
modified (e.g., without autoreactivity) with a sulfo-SE group and
used to label or otherwise modify biomolecules.
[0006] In some embodiments, provided herein are compositions
comprising a peptide linked to a sulfo n-hydroxysuccimidyl ester
(sulfo-SE) group, wherein the peptide does not comprise a cysteine
or lysine residue. In some embodiments, the sulfo-SE is linked to
the N-terminus of the peptide. In some embodiments, the sulfo-SE is
linked to the C-terminus of the peptide. In some embodiments, the
sulfo-SE is linked to an amino acid sidechain of the peptide. In
some embodiments, the peptide comprises at least one non-alkyl
amino acid selected from serine, threonine, tyrosine, glutamic
acid, arginine, histidine, tryptophan and aspartic acid. In some
embodiments, the at least one reactive non-alkyl amino acid is a or
tyrosine. In some embodiments, the at least one reactive
nucleophilic amino acid is an arginine. In some embodiments, the
sulfo-SE group is linked to the peptide by a non-peptide linker
group. In some embodiments, the linker group comprises and alkyl or
heteroalkyl chain. In some embodiments, the linker comprises one or
more sidechain substituents. In some embodiments, the peptide is
4-50 amino acids in length. In some embodiments, the peptide is
8-20 amino acids in length. In some embodiments, the sulfo-SE is
attached to the N-terminus of the peptide. In some embodiments, the
sulfo-SE is attached to the N-terminus of the peptide via a linker
group. In some embodiments, the peptides comprise a fluorophore or
chromophore conjugate. In some embodiments, the peptide is a
component of a biomolecular complex. In some embodiments, the
peptide is a component of a biomolecular complex. In some
embodiments, the peptide comprises 5 or fewer substitutions
relative to SEQ ID NO: 1 (SmBit). In some embodiments, one or more
lysines are replaced with an arginine. In some embodiments, one or
more lysines of SEQ ID NO: 1 are replaced with an arginine. In some
embodiments, the peptide comprises Pep691 (SEQ ID NO: 23). In some
embodiments, the peptide comprises SmBiT (SEQ ID NO: 10). In some
embodiments, the peptide is conjugated to a fluorophore. In some
embodiments, the peptide comprises fluorophore conjugated to an
arginine. In some embodiments, the peptide comprises fluorophore
conjugated to SEQ ID NO: 23. In some embodiments, the peptide
comprises fluorophore conjugated to SEQ ID NO: 10.
[0007] In some embodiments, provided herein are methods of labeling
a biomolecule with a peptide comprising contacting the biomolecule
with a sulfo-SE linked peptide described herein, under conditions
such that the sulfo-SE group reacts with an amine on the
biomolecule. In some embodiments, a peptide composition contacts
the biomolecule under conditions such that the sulfo-SE group
reacts with amine on the biomolecule. In some embodiments, the
amine is a primary amine. In some embodiments, the biomolecule is
selected from the group consisting of an antigen, an antibody, an
antibody fragment, a nanobody, a darpin, a non-antibody protein, a
receptor, a ligand, a toxin, a cytokine, a nucleic acid, a
nucleoprotein complex, a peptide, an amino acid, a sugar, a drug,
and streptavidin.
[0008] In some embodiments, provided herein are methods of labeling
a peptide with a sulfo-SE moiety comprising contacting the peptide
with a sulfo-NHS compound under conditions such that the hydroxy of
the sulfo-NHS compound reacts with the terminal amine of the
peptide, wherein the peptide does not comprise an cysteine or
lysine residue. In some embodiments, the peptide comprises at least
one reactive nucleophilic amino acid.
[0009] In some embodiments, provided herein are compositions
comprising a biomolecule labeled with a sulfo-SE linked peptide
described herein.
[0010] In some embodiments, provided herein are methods comprising
contacting comprising a biomolecule labeled with a sulfo-SE linked
peptide described herein with an analyte. In some embodiments, the
analyte is selected from the group consisting of an antigen, an
antibody, an antibody fragment, a nanobody, a darpin, a
non-antibody protein, a receptor, a ligand, a toxin, a cytokine, a
nucleic acid, a nucleoprotein complex, a peptide, an amino acid, a
sugar, a drug, and streptavidin. In some embodiments, the analyte
is linked to a complementary polypeptide capable of forming a
bioluminescent complex with the peptide on the biomolecule. In some
embodiments, methods further comprise contacting the bioluminescent
complex with a substrate for the bioluminescent complex and
detecting luminescence.
[0011] In some embodiments, provided herein are compositions
comprising an analyte labeled with a sulfo-SE linked peptide
described herein.
[0012] In some embodiments, provided herein are methods comprising
contacting an analyte labeled with a sulfo-SE linked peptide
described herein with a biomolecule. In some embodiments, the
biomolecule is linked to a complementary polypeptide capable of
forming a bioluminescent complex with the peptide on the analyte.
In some embodiments, methods further comprise contacting the
bioluminescent complex with a substrate for the bioluminescent
complex and detecting luminescence, fluorescence, and/or BRET.
[0013] In some embodiments, provided herein are compositions
comprising an analyte labeled with a first sulfo-SE linked peptide
described herein and a biomolecule labelled with a second sulfo-SE
linked peptide described herein, wherein the first and second
peptides are capable of forming a bioluminescent complex in the
presence of a complementary polypeptide. In some embodiments,
methods comprise contacting the analyte and biomolecule with the
complementary polypeptide and forming the bioluminescent complex.
In some embodiments, methods further comprise contacting the
bioluminescent complex with a substrate for the bioluminescent
complex and detecting luminescence.
[0014] In some methods herein, one or more of the peptides is a
fluorophore or chromophore-conjugated peptide. These methods
further comprise detecting fluorescence/light and/or BRET from a
bioluminescent complex to the fluorophore or chromophore. In some
embodiments, the number of labeling per biomolecule is calculated
by the number of fluorophore or chromophore molecules per
biomolecule. In some embodiments, the fluorophore molecule is a
FAM, TAMRA, ROX, silo-rhodamine, BODIPY, TOM, Dyomics dye, or a
carbon-rhodamine, but not limited to those fluorophores.
[0015] In some embodiments, provided herein are methods comprising
(a) forming a bioluminescent complex of a SEQ ID NO: 1 (SmBiT)
labeled-analyte biomolecule and the LgBiT-labeled analyte
biomolecule specific antibody; (b) contacting the bioluminescent
complex with the analyte; (c) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; and (d) detecting
light output from the bioluminescent complex.
[0016] In some embodiments, provided herein are methods comprising
(a) contacting an analyte with a SEQ ID NO: 1 (SmBiT)
labeled-analyte specific antibody and a LgBiT-labeled
analyte-specific antibody and forming a bioluminescent complex; (b)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (c) detecting light output from the
bioluminescent complex.
[0017] In some embodiments, provided herein are methods comprising
(a) contacting an analyte with a SEQ ID NO: 1 (SmBiT)
labeled-analyte specific antibody, a SEQ ID NO: 11 (HiBiT)-labeled
analyte-specific antibody, and a polypeptide copable for forming a
bioluminescent complex with HiBiT and SmBiT; (b) contacting the
bioluminescent complex with a substrate for the bioluminescent
complex; and (c) detecting light output from the bioluminescent
complex.
[0018] In some embodiments, provided herein are methods comprising:
(a) contacting an analyte with a SEQ ID NO: 1 (SmBiT)
labeled-analyte specific antibody, a SEQ ID NO: 11 (HiBiT)-labeled
analyte-specific antibody, and a polypeptide capable for forming a
bioluminescent complex with HiBiT and SmBiT; (b) contacting the
bioluminescent complex with a substrate for the bioluminescent
complex; (c) detecting light output from the bioluminescent
complex.
[0019] In some embodiments, provided herein are methods comprising
contacting an analyte with a biomolecule labeled with a sulfo-SE
linked peptide described herein. In some embodiments, the analyte
is selected from the group consisting of an antigen, an antibody, a
non-antibody protein, a receptor, a ligand, a toxin, a cytokine, a
nucleic acid, a peptide, an amino acid, a sugar, a drug, a
nucleoprotein complex, biotin, and streptavidin. In some
embodiments, the analyte biomolecule is labeled with SEQ ID NO: 1
(SmBiT).
[0020] In some embodiments, methods comprise: (a) forming a
bioluminescent complex from a SEQ ID NO: 1 (SmBiT) labeled-analyte
biomolecule and a LgBiT-labeled analyte biomolecule specific
antibody; (b) contacting the bioluminescent complex with the
analyte; (c) contacting the bioluminescent complex with a substrate
for the bioluminescent complex; and (d) detecting light output from
the bioluminescent complex.
[0021] In some embodiments, methods comprise: (a) forming a
bioluminescent complex from SEQ ID NO: 1 (SmBiT) labeled- and
LgBiT-labeled analyte specific antibodies; (b) contacting the
bioluminescent complex with the analyte; (c) contacting the
bioluminescent complex with a substrate for the bioluminescent
complex; and (d) detecting light output from the bioluminescent
complex.
[0022] In some embodiments, methods comprise: (a) contacting an
analyte with SEQ ID NO: 10 (SmBiT)-labeled antibodies or receptors
and SEQ ID NO: 11 (HiBiT)-labeled antibodies or receptors; (b)
contacting the analyte with LgBiT to form a bioluminescent complex;
(c) contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (d) detecting light output from the
bioluminescent complex.
[0023] In some embodiments, methods comprise: (a) contacting SmBiT-
or HiBiT-labeled-analyte biomolecule with a HiBiT- or SmBiT-labeled
analyte biomolecule specific antibody; (b) contacting with LgBiT to
form a bioluminescent complex; (c) contacting with the analyte; (d)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (e) detecting light output from the
bioluminescent complex. In some embodiments, the peptide is a
fluorophore or chromophore-conjugated peptide. In some embodiments,
the number of labeling per biomolecule is calculated by the number
of fluorophore or chromophore molecules per biomolecule. In some
embodiments, the fluorophore molecule is a FAM, TAMRA, ROX,
silo-rhodamine, BODIPY, TOM, Dyomics dye, or a carbon-rhodamine,
but not limited to those fluorophores.
[0024] In some embodiments, methods comprise: (a) forming a
bioluminescent complex from a fluorophore-conjugated SEQ ID NO: 1
(SmBiT) labeled-analyte biomolecule and a LgBiT-labeled analyte
biomolecule specific antibody; (b) contacting the bioluminescent
complex with the analyte; (c) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; and (d) detecting
light output from the bioluminescent complex.
[0025] In some embodiments, methods comprise: (a) contacting an
analyte with both a fluorophore-conjugated SEQ ID NO: 1 (SmBiT)
labeled- and LgBiT-labeled analyte specific antibodies, forming a
bioluminescent complex; (b) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; (c) detecting
light output from the bioluminescent complex.
[0026] In some embodiments, methods comprise: (a) one of SEQ ID NO:
10 (SmBiT) and SEQ ID NO: 11 (HiBiT)-peptides is the
fluorophore-conjugated peptide; (b) the analyte contacts SEQ ID NO:
10 (SmBiT) and SEQ ID NO: 11 (HiBiT)-labeled antibodies, or
receptors, or combination, wherein one of the peptides is a
fluorophore-conjugated peptide; (b) contacting with LgBiT to form a
bioluminescent complex; (c) contacting the bioluminescent complex
with a substrate for the bioluminescent complex; and (d) detecting
light output from the bioluminescent complex.
[0027] In some embodiments, provided herein are methods wherein the
analyte biomolecule or analyte specific antibody is labeled with
fluorophore-conjugated SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11
(HiBiT), wherein (a) SEQ ID NO: 10 (SmBiT) or SEQ ID NO: 11
HiBiT.labeled-analyte biomolecule contacts the SEQ ID NO: 11
(HiBiT) or SEQ ID NO: 10 (SmBiT)-labeled analyte biomolecule
specific antibody, wherein one of the peptides is the
fluorophore-conjugated peptide; (b) contacting with LgBiT to form a
bioluminescent complex; (c) contacting with the analyte; (d)
contacting the bioluminescent complex with a substrate for the
bioluminescent complex; and (e) detecting light output from the
bioluminescent complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A-B. Synthetic schemes for attaching a generic
sulfo-SE group to a generic peptide: (A) the target peptide was
reacted directly with bis-sulfo-SE: one of the sulfo-SE reacts with
free amine on the N-terminus, C-terminus or side chain while
leaving the other sulfo-SE intact); (B) the protected peptide on
resin is first coupled with bis-Sulfo SE at either N-terminus or
side chain, and the resulting peptide was then cleaved off resin in
trifluoroacetic acid.
[0029] FIG. 2. Synthetic scheme for attaching a sulfo-SE peptide to
a generic biomolecule.
[0030] FIG. 3A-B. Attachment of the SmBiT peptide to a target of
interest via (A) HALOTAG and (B) biotin/streptavidin systems.
[0031] FIG. 4. SmBiT-Sulfo-SE labeling of an antigen.
[0032] FIG. 5. Evaluation of SE-SmBiT-labeled Goat anti-mouse
antibody in a direct immunoassay.
[0033] FIG. 6. Evaluation of SE-SmBiT-labeled Streptavidin in an
IFN.gamma. indirect immunoassay.
[0034] FIG. 7. Evaluation of SE-SmBiT-labeled Streptavidin in a
fumonisin competition immunoassay.
[0035] FIG. 8. Evaluation of SE-SmBiT-labeled Streptavidin in an
FcRn binding assay.
[0036] FIG. 9. Schematic illustration depicting exemplary
bioluminescent immunoassay using components and reagents described
herein.
[0037] FIG. 10A-B. Graphs depicting bioluminescent quantitation of
human recombinant IL-6 using Sulfo-SE-SmTrip9 (824) (SEQ ID NO: 25)
and Sulfo-SE-SmTrip10 (691) (SEQ ID NO: 23) directly labeled paired
monoclonal antibodies combined with LgTrip (3546) (SEQ ID NO: 12)
and furimazine in a solution based homogeneous assay. A) raw RLU
values and B) signal to noise calculations.
[0038] FIG. 11A-B. Graphs depicting bioluminescent quantitation of
human recombinant IL-6 using Sulfo-SE-PEG3-SmTrip9 (693) (SEQ ID
NO: 16) or Sulfa-SE-PEG3-SmTrip9 (840) (SEQ ID NO: 17) and
Sulfo-SE-PEG3-SmTrip10 (691) (SEQ ID NO: 23) directly labeled
paired monoclonal antibodies combined with LgTrip (3546) (SEQ ID
NO: 12) and furimazine in a solution based homogeneous assay. A)
raw RLU values and B) signal to noise calculations.
[0039] FIG. 12. Schematic illustration depicting exemplary
bioluminescent resonance energy transfer (NanoBRET) immunoassay
with the donor light generation resulting from complementation of
the NanoTrip system using components and reagents described
herein.
[0040] FIG. 13. Graph depicting real time binding kinetics for
NanoBRET based quantitation of human recombinant IL-6 using
Sulfo-SE-PEG3-SmTrip9(938)-TAMRA (SEQ ID NO: 38) and
Sulfo-SE-PEG3-SmTrip10(691) (SEQ ID NO: 23) directly labeled paired
monoclonal antibodies with LgTrip (3546) (SEQ ID NO: 12) and
furimazine in a solution-based homogeneous assay.
[0041] FIG. 14A-B. SDS PAGE gels of directly labeled anti-human
IL-6 monoclonal antibodies. A) brightfield image capturing total
protein and B) fluorescent image capturing Sulfo-SE-peptide-TAMRA
(labeled antibody and excess label).
[0042] FIG. 15. Exemplary sulfo-SE peptides.
DEFINITIONS
[0043] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments described herein, some preferred methods, compositions,
devices, and materials are described herein. However, before the
present materials and methods are described, it is to be understood
that this invention is not limited to the particular molecules,
compositions, methodologies or protocols herein described, as these
may vary in accordance with routine experimentation and
optimization. It is also to be understood that the terminology used
in the description is for the purpose of describing the particular
versions or embodiments only and is not intended to limit the scope
of the embodiments described herein.
[0044] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. However,
in case of conflict, the present specification, including
definitions, will control. Accordingly, in the context of the
embodiments described herein, the following definitions apply.
[0045] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
"a peptide" is a reference to one or more peptides and equivalents
thereof known to those skilled in the art, and so forth.
[0046] As used herein, the term "and/or" includes any and all
combinations of listed items, including any of the listed items
individually. For example, "A, B, and/or C" encompasses A, B, C,
AB, AC, BC, and ABC, each of which is to be considered separately
described by the statement "A, B, and/or C."
[0047] As used herein, the term "comprise" and linguistic
variations thereof denote the presence of recited feature(s),
element(s), method step(s), etc. without the exclusion of the
presence of additional feature(s), element(s), method step(s), etc.
Conversely, the term "consisting of" and linguistic variations
thereof, denotes the presence of recited feature(s), element(s),
method step(s), etc. and excludes any unrecited feature(s),
element(s), method step(s), etc., except for ordinarily-associated
impurities. The phrase "consisting essentially of" denotes the
recited feature(s), element(s), method step(s), etc. and any
additional feature(s), element(s), method step(s), etc. that do not
materially affect the basic nature of the composition, system, or
method. Many embodiments herein are described using open
"comprising" language. Such embodiments encompass multiple closed
"consisting of" and/or "consisting essentially of" embodiments,
which may alternatively be claimed or described using such
language.
[0048] As used herein, the term "substantially" means that the
recited characteristic, parameter, and/or value need not be
achieved exactly, but that deviations or variations, including for
example, tolerances, measurement error, measurement accuracy
limitations and other factors known to skill in the art, may occur
in amounts that do not preclude the effect the characteristic was
intended to provide. A characteristic or feature that is
substantially absent (e.g., substantially non-luminescent) may be
one that is within the noise, beneath background, below the
detection capabilities of the assay being used, or a small fraction
(e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%,
<0.000001%, <0.0000001%) of the significant characteristic
(e.g., luminescent intensity of a bioluminescent protein or
bioluminescent complex).
[0049] As used herein, the term "sulfo n-hydroxysuccinimidyl ester"
("sulfo-SE") refers to a moiety on a chemical or biomolecule having
the chemical structure:
##STR00001##
[0050] wherein R is the chemical or biomolecule (e.g., peptide) and
L is any suitable linker (as described herein) connecting the
sulfo-SE group to the chemical or biomolecule (e.g., peptide).
[0051] The term "amino acid" refers to natural amino acids,
unnatural amino acids, and amino acid analogs, all in their D and L
stereoisomers, unless otherwise indicated, if their structures
allow such stereoisomeric forms.
[0052] Natural amino acids include alanine (Ala or A), arginine
(Arg or R), asparagine (Asn or N), aspartic acid (Asp or D),
cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or
E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or
I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M),
phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S),
threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y)
and valine (Val or V).
[0053] Unnatural amino acids include, but are not limited to,
pentafluorophenylalanine ("Z"), azetidinecarboxylic acid,
2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,
naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid,
4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,
2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic
acid, tertiary-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid,
desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,
N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"),
hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"),
4-hydroxyproline ("4Hyp"), isodemosine, allo-isoleucine,
N-methylalanine ("MeAla" or "Nime"), N-alkylglycine ("NAG")
including N-methylglycine, N-methylisoleucine, N-alkylpentylglycine
("NAPG") including N-methylpentylglycine. N-methylvaline,
naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"),
octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or
"PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"),
homoLysine ("hLys"), citruline, halo-substituted tyrosine, and
homoArginine ("hArg"). Unnatural reactive amino acids are described
in, for example, Boutureira, O. and G. J. Bernardes (2015)
"Advances in chemical protein modification." Chem Rev 115(5):
2174-2195; herein incorporated by reference in its entirety.
[0054] The term "amino acid analog" refers to a natural or
unnatural amino acid where one or more of the C-terminal carboxy
group, the N-terminal amino group and side-chain bioactive group
has been chemically blocked, reversibly or irreversibly, or
otherwise modified to another bioactive group. For example,
aspartic acid-(beta-methyl ester) is an amino acid analog of
aspartic acid; N-ethylglycine is an amino acid analog of glycine;
or alanine carboxamide is an amino acid analog of alanine. Other
amino acid analogs include methionine sulfoxide, methionine
sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine
sulfoxide and S-(carboxymethyl)-cysteine sulfone. Amino acid
analogs may comprise amino acids with various protecting groups
(Isidro-Llobet, A., et al. (2009). "Amino Acid-Protecting Groups."
Chemical Reviews 109(6): 2455-2504; herein incorporated by
reference in its entirety).
[0055] As used herein, unless otherwise specified, the terms
"peptide" and "polypeptide" refer to polymer compounds of two or
more amino acids joined through the main chain by peptide amide
bonds (--C(O)NH--). The term "peptide" typically refers to short
amino acid polymers (e.g., chains having fewer than 50 amino
acids), whereas the term "polypeptide" typically refers to longer
amino acid polymers (e.g., chains having more than 50 amino
acids).
[0056] As used herein, terms "peptidomimetic" and "peptide mimetic"
refer to peptide-like or polypeptide-like molecules that emulate a
sequence derived from a protein or peptide. A peptidomimetic may
contain amino acids analogs, peptoid amino acids, and/or non-amino
acid components either exclusively or in combination with amino
acids (e.g., natural or non-natural amino acids). Examples of
peptidomimetics include chemically modified peptides/polypeptides,
peptoids (side chains are appended to the nitrogen atom of the
peptide backbone rather than to the .alpha.-carbons),
.beta.-peptides (amino group bonded to the .beta. carbon rather
than the .alpha. carbon), etc.
[0057] As used herein, the term "peptoid" refers to a class of
peptidomimetics where the side chains are functionalized on the
nitrogen atom of the peptide backbone rather than to the
.alpha.-carbon.
[0058] As used herein, the term "artificial" refers to compositions
and systems that are designed or prepared by man and are not
naturally occurring. For example, an artificial peptide, peptoid,
or nucleic acid is one comprising a non-natural sequence (e.g., a
peptide without 100% identity with a naturally-occurring protein or
a fragment thereof).
[0059] As used herein, a "conservative" amino acid substitution
refers to the substitution of an amino acid in a peptide or
polypeptide with another amino acid having similar chemical
properties such as size or charge. For purposes of the present
disclosure, each of the following eight groups contains amino acids
that are conservative substitutions for one another: [0060] 1)
Alanine (A) and Glycine (G); [0061] 2) Aspartic acid (D) and
Glutamic acid (E); [0062] 3) Asparagine (N) and Glutamine (Q);
[0063] 4) Arginine (R) and Lysine (K); [0064] 5) Isoleucine (I),
Leucine (L), Methionine (M), and Valine (V); [0065] 6)
Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); [0066] 7)
Serine (S) and Threonine (T); and [0067] 8) Cysteine (C) and
Methionine (M).
[0068] Naturally occurring residues may be divided into classes
based on common side chain properties, for example: polar positive
(or basic) (histidine (H), lysine (K), and arginine (R)); polar
negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar
neutral (serine (S), threonine (T), asparagine (N), glutamine (Q));
non-polar aliphatic (alanine (A), valine (V), leucine (L),
isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine
(F), tyrosine (Y), tryptophan (W)); proline and glycine; and
cysteine. As used herein, a "semi-conservative" amino acid
substitution refers to the substitution of an amino acid in a
peptide or polypeptide with another amino acid within the same
class.
[0069] In some embodiments, unless otherwise specified, a
conservative or semi-conservative amino acid substitution may also
encompass non-naturally occurring amino acid residues that have
similar chemical properties to the natural residue. These
non-natural residues are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems. These
include, but are not limited to, peptidomimetics and other reversed
or inverted forms of amino acid moieties. Embodiments herein may,
in some embodiments, be limited to natural amino acids, non-natural
amino acids, and/or amino acid analogs.
[0070] Non-conservative substitutions may involve the exchange of a
member of one class for a member from another class.
[0071] As used herein, the term "sequence identity" refers to the
degree two polymer sequences (e.g., peptide, polypeptide, nucleic
acid, etc.) have the same sequential composition of monomer
subunits. The term "sequence similarity" refers to the degree with
which two polymer sequences (e.g., peptide, polypeptide, nucleic
acid, etc.) have similar polymer sequences. For example, similar
amino acids are those that share the same biophysical
characteristics and can be grouped into the families, e.g., acidic
(e.g., aspartate, glutamate), basic (e.g., lysine, arginine,
histidine), non-polar (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan) and uncharged polar
(e.g., glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine). The "percent sequence identity" (or "percent sequence
similarity") is calculated by: (1) comparing two optimally aligned
sequences over a window of comparison (e.g., the length of the
longer sequence, the length of the shorter sequence, a specified
window), (2) determining the number of positions containing
identical (or similar) monomers (e.g., same amino acids occurs in
both sequences, similar amino acid occurs in both sequences) to
yield the number of matched positions, (3) dividing the number of
matched positions by the total number of positions in the
comparison window (e.g., the length of the longer sequence, the
length of the shorter sequence, a specified window), and (4)
multiplying the result by 100 to yield the percent sequence
identity or percent sequence similarity. For example, if peptides A
and B are both 20 amino acids in length and have identical amino
acids at all but 1 position, then peptide A and peptide B have 95%
sequence identity. If the amino acids at the non-identical position
shared the same biophysical characteristics (e.g., both were
acidic), then peptide A and peptide B would have 100% sequence
similarity. As another example, if peptide C is 20 amino acids in
length and peptide D is 15 amino acids in length, and 14 out of 15
amino acids in peptide D are identical to those of a portion of
peptide C, then peptides C and D have 70% sequence identity, but
peptide D has 93.3% sequence identity to an optimal comparison
window of peptide C. For the purpose of calculating "percent
sequence identity" (or "percent sequence similarity") herein, any
gaps in aligned sequences are treated as mismatches at that
position.
[0072] Any peptide/polypeptides described herein as having a
particular percent sequence identity or similarity (e.g., at least
70%) with a reference sequence ID number, may also be expressed as
having a maximum number of substitutions (or terminal deletions)
with respect to that reference sequence. For example, a sequence
having at least Y % sequence identity (e.g., 90%) with SEQ ID NO:Z
(e.g., 100 amino acids) may have up to X substitutions (e.g., 10)
relative to SEQ ID NO:Z, and may therefore also be expressed as
"having X (e.g., 10) or fewer substitutions relative to SEQ ID
NO:Z."
[0073] As used herein, the term "sample" is used in its broadest
sense. In one sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from animals (including
humans) and encompass fluids, solids, tissues, and gases.
Biological samples include blood products, such as plasma, serum,
and the like. Sample may also refer to cell lysates or purified
forms of the enzymes, peptides, and/or polypeptides described
herein. Cell lysates may include cells that have been lysed with a
lysing agent or lysates such as rabbit reticulocyte or wheat germ
lysates. Sample may also include cell-free expression systems.
Environmental samples include environmental material such as
surface matter, soil, water, crystals, and industrial samples. Such
examples are not however to be construed as limiting the sample
types applicable to the present invention.
[0074] As used herein, the term "physiological conditions"
encompasses any conditions compatible with living cells, e.g.,
predominantly aqueous conditions of a temperature, pH, salinity,
chemical makeup, etc. that are compatible with living cells.
[0075] As used herein, the terms "conjugated" and "conjugation"
refer to the covalent attachment of two molecular entities (e.g.,
post-synthesis and/or during synthetic production). The attachment
of a peptide or small molecule tag to a protein or small molecule,
chemically (e.g., "chemically" conjugated) or enzymatically, is an
example of conjugation. The reaction of a SE-peptide with an amine
on a biomolecule results in conjugation of the peptide with the
biomolecule.
[0076] The term "binding moiety" refers to a domain that
specifically binds an antigen or epitope independently of a
different epitope or antigen binding domain. A binding moiety may
be an antibody, antibody fragment, a receptor domain that binds a
target ligand, proteins that bind to immunoglobulins (e.g., protein
A, protein G, protein A/G, protein L, protein M), a binding domain
of a proteins that bind to immunoglobulins (e.g., protein A,
protein G, protein A/G, protein L, protein M), oligonucleotide
probe, peptide nucleic acid, DARPin, aptamer, affimer, a purified
protein (either the analyte itself or a protein that binds to the
analyte), and analyte binding domain(s) of proteins etc. Table A
provides a list of exemplary binding moieties that could be used
singly or in various combinations in methods, systems, and assays
(e.g., immunoassays) herein.
TABLE-US-00001 TABLE A Exemplary binding moieties Protein A Ig
Binding domain of protein A Protein G Ig Binding domain of protein
G Protein L Ig Binding domain of protein L Protein M Ig Binding
domain of protein M polyclonal antibody against analyte X
monoclonal antibody recombinant antibody scFv variable light chain
(V.sub.L) of antibody (monoclonal, recombinant, polyclonal)
recognizing target analyte X protein (e.g. receptor) binding domain
that binds to analyte X (Fab) fragment Fab' fragment Fv fragment
F(ab')2 fragment oligonucleotide probe DARPins and other synthetic
binding scaffolds (ex: Bicycles) peptide nucleic acid aptamer
affimer
[0077] As used herein, the term "antibody" refers to a whole
antibody molecule or a fragment thereof (e.g., fragments such as
Fab, Fab', and F(ab').sub.2, variable light chain, variable heavy
chain, Fv, it may be a polyclonal or monoclonal or recombinant
antibody, a chimeric antibody, a humanized antibody, a human
antibody, etc. As used herein, when an antibody or other entity
"specifically recognizes" or "specifically binds" an antigen or
epitope, it preferentially recognizes the antigen in a complex
mixture of proteins and/or macromolecules and binds the antigen or
epitope with affinity which is substantially higher than to other
entities not displaying the antigen or epitope. In this regard,
"affinity which is substantially higher" means affinity that is
high enough to enable detection of an antigen or epitope which is
distinguished from entities using a desired assay or measurement
apparatus. Typically, it means binding affinity having a binding
constant (K.sub.a) of at least 10.sup.7 M.sup.-1 (e.g.,
>10.sup.7 M.sup.-1, >10.sup.8 M.sup.-1, >10.sup.9
M.sup.-1, >10.sup.10 M.sup.-1, >10.sup.11 M.sup.-1,
>10.sup.12 M.sup.-1, >10.sup.13 M.sup.-1, etc.). In certain
such embodiments, an antibody is capable of binding different
antigens so long as the different antigens comprise that particular
epitope. In certain instances, for example, homologous proteins
from different species may comprise the same epitope.
[0078] As used herein, the term "antibody fragment" refers to a
portion of a full-length antibody, including at least a portion of
the antigen binding region or a variable region. Antibody fragments
include, but are not limited to, Fab, Fab', F(ab').sub.2, Fv, scFv,
Fd, variable light chain, variable heavy chain, diabodies, and
other antibody fragments that retain at least a portion of the
variable region of an intact antibody. See, e.g., Hudson et al.
(2003) Nat. Med. 9:129-134; herein incorporated by reference in its
entirety. In certain embodiments, antibody fragments are produced
by enzymatic or chemical cleavage of intact antibodies (e.g.,
papain digestion and pepsin digestion of antibody) produced by
recombinant DNA techniques, or chemical polypeptide synthesis. For
example, a "Fab" fragment comprises one light chain and the
C.sub.H1 and variable region of one heavy chain. The heavy chain of
a Fab molecule cannot form a disulfide bond with another heavy
chain molecule. A "Fab'" fragment comprises one light chain and one
heavy chain that comprises an additional constant region extending
between the C.sub.H1 and C.sub.H2 domains. An interchain disulfide
bond can be formed between two heavy chains of a Fab' fragment to
form a "F(ab')2" molecule. An "Fv" fragment comprises the variable
regions from both the heavy and light chains, but lacks the
constant regions. A single-chain Fv (scFv) fragment comprises heavy
and light chain variable regions connected by a flexible linker to
form a single polypeptide chain with an antigen-binding region.
Exemplary single chain antibodies are discussed in detail in WO
88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203; herein
incorporated by reference in their entireties. In certain
instances, a single variable region (e.g., a heavy chain variable
region or a light chain variable region) may have the ability to
recognize and bind antigen. Other antibody fragments will be
understood by skilled artisans.
[0079] As used herein, the term "peptide tag" refers to a peptide
that may be attached (e.g., post-synthesis or during synthetic
production) or fused to another entity (e.g., a biomolecule). In
typical embodiments herein, a peptide tag displays (is linked to) a
sulfo-SE group (e.g., for conjugation of the peptide to a
biomolecule). In certain embodiments herein, a peptide tag is
capable of forming a bioluminescent complex with (i) a polypeptide
and/or (ii) another peptide tag and a polypeptide, under
appropriate conditions.
[0080] As used herein, the term "biomolecule" or "biological
molecule" is refer to molecules and ions that are present in
organisms and are essential to a biological process(es) such as
cell division, morphogenesis, or development. [1] Biomolecules
include large macromolecules (or polyanions) such as proteins,
carbohydrates, lipids, and nucleic acids as well as small molecules
such as primary metabolites, secondary metabolites, and natural
products. A more general name for this class of material is
biological materials. Biomolecules are usually endogenous, but may
also be exogenous. For example, pharmaceutical drugs may be natural
products or semisynthetic (biopharmaceuticals), or they may be
totally synthetic.
[0081] As used herein, the term "bioluminescence" refers to
production and emission of light by a chemical reaction catalyzed
by, or enabled by, an enzyme, protein, protein complex, or other
biomolecule (e.g., bioluminescent complex). In typical embodiments,
a substrate for a bioluminescent entity (e.g., bioluminescent
protein or bioluminescent complex) is converted into an unstable
form by the bioluminescent entity; the substrate subsequently emits
light. As used herein, the term "non-luminescent" refers to an
entity (e.g., peptide, polypeptide, complex, protein, etc.) that
exhibits the characteristic of not emitting a detectable amount of
light in the visible spectrum (e.g., in the presence of a
substrate). For example, an entity may be referred to as
non-luminescent if it does not exhibit detectable luminescence in a
given assay. As used herein, the term "non-luminescent" is
synonymous with the term "substantially non-luminescent. In some
embodiments, an entity is considered "non-luminescent" if any light
emission is sufficiently minimal so as not to create interfering
background for a particular assay.
[0082] As used herein, the terms "non-luminescent peptide" and
"non-luminescent polypeptide" refer to peptides and polypeptides
that exhibit substantially no luminescence (e.g., in the presence
of a substrate), or an amount that is beneath the noise (e.g.,
100-fold, 200-fold, 500-fold, 1.times.10.sup.3-fold,
1.times.10.sup.4-fold, 1.times.10.sup.5-fold,
1.times.10.sup.6-fold, 1.times.10.sup.7-fold, etc.), when compared
to a significant signal (e.g., a bioluminescent complex) under
standard conditions (e.g., physiological conditions, assay
conditions, etc.) and with typical instrumentation (e.g.,
luminometer, etc.). In some embodiments, such non-luminescent
peptides and polypeptides assemble, according to the criteria
described herein, to form a bioluminescent complex.
[0083] As used herein, the term "an Oplophorus luciferase" ("an
OgLuc") refers to a luminescent polypeptide having significant
sequence identity, structural conservation, and/or the functional
activity of the luciferase produce by and derived from the deep-sea
shrimp Oplophorus gracilirostris. In particular, an OgLuc
polypeptide refers to a luminescent polypeptide having significant
sequence identity, structural conservation, and/or the functional
activity of the mature 19 kDa subunit of the Oplophorus luciferase
protein complex (e.g., without a signal sequence) such as SEQ ID
NOs: 1 (WT OgLuc) and 3 (NanoLuc), which comprises 10 .beta.
strands (.beta.1, .beta.2, .beta.3, .beta.4, .beta.5, .beta.6,
.beta.7, .beta.8, .beta.9, .beta.10) and utilize substrates such as
coelenterazine or a coelenterazine derivative or analog to produce
luminescence.
[0084] As used herein the term "complementary" refers to the
characteristic of two or more structural elements (e.g., peptide,
polypeptide, nucleic acid, small molecule, etc.) of being able to
hybridize, dimerize, or otherwise form a complex with each other.
For example, a "complementary peptide and polypeptide" are capable
of coming together to form a complex. Complementary elements may
require assistance (facilitation) to form a complex (e.g., from
interaction elements), for example, to place the elements in the
proper conformation for complementarity, to place the elements in
the proper proximity for complementarity, to co-localize
complementary elements, to lower interaction energy for
complementary, to overcome insufficient affinity for one another,
etc.
[0085] As used herein, the term "complex" refers to an assemblage
or aggregate of molecules (e.g., peptides, polypeptides, etc.) in
direct and/or indirect contact with one another. In one aspect,
"contact," or more particularly, "direct contact" means two or more
molecules are close enough so that attractive noncovalent
interactions, such as Van der Waal forces, hydrogen bonding, ionic
and hydrophobic interactions, and the like, dominate the
interaction of the molecules. In such an aspect, a complex of
molecules (e.g., peptides and polypeptide) is formed under assay
conditions such that the complex is thermodynamically favored
(e.g., compared to a non-aggregated, or non-complexed, state of its
component molecules). As used herein the term "complex," unless
described as otherwise, refers to the assemblage of two or more
molecules (e.g., peptides, polypeptides or a combination
thereof).
[0086] As used herein, the term ".beta.9-like peptide" refers to a
peptide (or peptide tag) comprising significant sequence identity,
structural conservation, and/or the functional activity of the
.beta. (beta) 9 strand of an OgLuc polypeptide. In particular, a
.beta.9-like peptide is a peptide capable of structurally
complementing an OgLuc polypeptide lacking a .beta.9 strand
resulting in enhanced luminescence of the complex compared to the
OgLuc polypeptide in the absence of the .beta.9-like peptide. Other
".beta.X-like peptides" may be similarly named (e.g., .beta.1-like,
.beta.2-like, .beta.3-like, .beta.4-like, .beta.5-like,
.beta.6-like, .beta.7-like, .beta.8-like, .beta.9-like). In some
embodiments, a .beta.9-like peptide is linked to a sulfo-SE group
for conjugation to a biomolecule.
[0087] As used herein, the term ".beta.10-like peptide" refers to a
peptide (or peptide tag) comprising significant sequence identity,
structural conservation, and/or the functional activity of the
.beta. (beta) 10 strand of an OgLuc polypeptide. In particular, a
.beta.10-like peptide is a peptide capable of structurally
complementing an OgLuc polypeptide lacking a .beta.10 strand
resulting in enhanced luminescence of the complex compared to the
OgLuc polypeptide in the absence of the .beta.10-like peptide.
Other ".beta.X-like peptides" may be similarly named (e.g.,
.beta.1-like, .beta.2-like, .beta.3-like, .beta.4-like,
.beta.5-like, .beta.6-like, .beta.7-like, .beta.8-like,
.beta.9-like). In some embodiments, a .beta.10-like peptide is
linked to a sulfo-SE group for conjugation to a biomolecule.
[0088] As used herein, the term ".beta..sub.1-8-like polypeptide"
refers to a polypeptide bearing sequence and structural similarity
to .beta. (beta) strands 1-8 of an OgLuc polypeptide, but lacking
.beta. (beta) strands 9 and 10. Other ".beta..sub.y-z-like
polypeptides" may be similarly named (e.g., .beta.14-like
polypeptide, .beta..sub.2-8-like polypeptide, .beta..sub.5-10-like
polypeptide, etc.).
[0089] As used herein, the term "NANOLUC" refers to an artificial
luciferase or bioluminescent polypeptide produced commercially by
the Promega Corporation and corresponding to SEQ ID NO: 3.
[0090] As used herein, the term "LgBiT" refers to a polypeptide
corresponding to .beta..sub.1-9-like polypeptide that finds use in,
for example, binary complementation to form a bioluminescent
complex and corresponds to SEQ ID NO: 11.
[0091] As used herein, the term "SmBiT" refers to a peptide
corresponding to .beta..sub.10-like peptide that finds use in, for
example, binary complementation to form a bioluminescent complex,
but has low affinity for LgBiT (e.g., requires facilitation for
complex formation) and corresponds to SEQ ID NO: 13. In some
embodiments, SmBit, or variants thereof, is linked to a sulfo-SE,
for conjugation to a biomolecule.
[0092] As used herein, the term "HiBiT" refers to a peptide
corresponding to .beta..sub.10-like peptide that finds use in, for
example, binary complementation to form a bioluminescent complex,
but has low affinity for LgBiT (e.g., requires facilitation for
complex formation). An exemplary HiBiT peptide corresponds to SEQ
ID NO: 15. HiBiT is has the same sequence as "SmHiTrip10" (SEQ ID
NO: 11) and "pep86," terms which may be used interchangeably (also
SmTrip10 pep86, etc.). In some embodiments, HiBiT variants lacking
cysteine and lysine residues are linked to a sulfo-SE for
conjugation to a biomolecule.
[0093] As used herein, the term "LgTrip" refers to a polypeptide
corresponding to .beta..sub.1-8-like polypeptide. An exemplary
LgTrip corresponds to SEQ ID NO: 17 and finds use in, for example,
tripartite complementation with .beta..sub.9-like and
.beta..sub.10-like peptides to form a bioluminescent complex, or
binary complementation, with a .beta..sub.9-10-like dipeptide to
form a bioluminescent complex. LgTrip variants include: LgTrip 2098
(w/His tag: SEQ ID NO: 31; w/o His tag: SEQ ID NO: 304) and LgTrip
3546 (w/His tag: SEQ ID NO: 51; w/o His tag: SEQ ID NO: 302).
[0094] As used herein, the term "SmTrip10" refers to a peptide
corresponding to .beta..sub.10-like peptide that finds use in, for
example, tripartite complementation to form a bioluminescent
complex. In some embodiments, SmTrip10, or variants thereof, is
linked to a sulfo-SE, for conjugation to a biomolecule.
[0095] As used herein, the term "SmTrip9" refers to a peptide
corresponding to .beta..sub.9-like peptide that finds use in, for
example, tripartite complementation to form a bioluminescent
complex. In some embodiments, SmTrip9, or variants thereof, is
linked to a sulfo-SE, for conjugation to a biomolecule.
[0096] Various peptide and polypeptide sequences that find use in
certain embodiments herein are described in U.S. Pat. No. 9,797,889
(herein incorporated by reference in its entirety) and Intl. App.
No. PCT/US19/36844 (herein incorporated by reference in its
entirety).
DETAILED DESCRIPTION
[0097] Provided herein are sulfo n-hydroxysuccimidyl ester
(sulfo-SE) linked peptides, methods of synthesis thereof, and
methods of using such peptides for labeling of biomolecules. In
particular, peptides comprising a non-alkyl group(s) such as
serine, threonine, arginine, tyrosine, glutamic acid, and aspartic
acid are stably modified (e.g., without autoreactivity) with a
sulfo-SE group and used to label or otherwise modify
biomolecules.
[0098] Synthesizing sulfo-SE peptides (e.g., peptides displaying
sulfo-SE groups) is not trivial due to the high potential for
autoreactivity of SE groups with the peptides to which they are
attached. For example, the presence of amino acids with accessible
nucleophilic side chains (e.g., arginine, lysine, histidine,
cysteine, serine, tyrosine, etc.) on a peptide has been understood
by the field to result in potential autoreactivity of the SE-group
with such a residue. As such, peptide attachment to biomolecules
through SE reaction with exposed amines on biomolecules has not
been pursued. However, experiments conducted during development of
embodiments herein have demonstrated the successful attachment of
N-hydroxysulfosuccinimide (sulfo-NHS) to a peptide lacking lysine
and cysteine residues (but containing other nucleophilic residues)
resulting in the formation of a sulfo n-hydroxysuccinimidyl ester
(sulfo-SE) on the N-terminus of the peptide without subsequence
autoreactivity of the sulfo-SE group with the peptide. The sulfo-SE
labeled peptide was subsequently used to peptide-label biomolecules
(e.g., streptavidin, antibodies, etc.) via reaction of the sulfo-SE
group with primary amines on the biomolecules. These experiments
demonstrate that stable sulfo-SE peptides can be generated by the
reaction of sulfo-NHS with peptides (e.g., peptides lacking lysine
and cysteine amino acids) to produce useful reagents for the
peptide labeling of biomolecules.
[0099] In some embodiments, provided herein are methods for the
synthesis of sulfo-SE peptides (FIG. 1). In some embodiments, a
peptide is combined with bis-sulfo-SE (e.g., under appropriate
reaction conditions (e.g., in PBS buffer, pH 7.6)) to produce a
sulfo-SE-peptide (e.g., sulfo-SE/peptide conjugate). In some
embodiments, the bis-sulfo-SE reacts with the N-terminal amine of
the peptide on resin to result in an end-labeled peptide on resin
and it was cleaved off resin with trifluoroacetic acid. Experiments
conducted during development of embodiments herein demonstrate that
in the absence of lysine and cysteine residues in the peptide, the
sulfo-SE group will not autoreact with the side chains of the
peptide.
[0100] In some embodiments, a suitable peptide for addition to an
N-terminal sulfo-SE label is, for example, 4 to 50 amino acids in
length (e.g., 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35,
40, 45, 50, or ranges therebetween). In some embodiments, the
peptide lacks cysteine residues. In some embodiments, the peptide
lacks lysine residues. In some embodiments, the peptide comprises
one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or
ranges therebetween) amino acids with a non-alkyl group such as
histidine, arginine, serine, tyrosine, aspartic acid, glutamic
acid. In some embodiments, the peptide comprises one or more (e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or ranges therebetween)
tyrosine, arginine, and/or glutamic acid amino acids.
[0101] In some embodiments, provided herein are compositions for
attaching a sulfo-SE moiety to a suitable peptide. In some
embodiments, a bis-sulfo-SE compound is provided. In some
embodiments, provided herein are compounds having a chemical
structure of:
##STR00002##
wherein L is any suitable linker (as described herein). In some
embodiments, L is selected from a straight alkyl chain (e.g., 1-20
carbons), a branched alkyl chain (e.g., 1-20 carbons), a straight
heteroalkyl (e.g., O, N, or S atoms within the alkyl), a branched
heteroalkyl, a substituted alkyl (e.g., suitable functional groups
along the alkyl chain), substituted heteroalkyl, etc. In some
embodiments, the linker comprises a linear or branched, cyclic or
heterocyclic, saturated or unsaturated, structure having 1-20
nonhydrogen atoms (e.g., C, N, P, O and S) and is composed of any
combination of alkyl, ether, thioether, imine, carboxylic, amine,
ester, carboxamide, sulfonamide, hydrazide bonds, carbamate, and
aromatic or heteroaromatic bonds. In some embodiments, linkers are
longer than 20 nonhydrogen atoms (e.g. 21 non-hydrogen atoms, 25
non-hydrogen atoms, 30 non-hydrogen atoms, 40 non-hydrogen atoms,
50 non-hydrogen atoms, 100 non-hydrogen atoms, etc.) In some
embodiments, the linker comprises 1-50 non-hydrogen atoms (in
addition to hydrogen atoms) selected from the group of C, N, P, O
and S (e.g. 1, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or
50 non-hydrogen atoms). In some embodiments, a linker comprises a
combination of CH.sub.2, (CH.sub.2).sub.2O, and carbamate groups.
For example, a linked may comprise --OC(O)(CH.sub.2).sub.6C(O)--,
or any other suitable combination of the functional groups
described herein.
[0102] In some embodiments, provided herein are reactants or a
reaction mix comprising sulfo-NHS (with or without an R linker
group) and a suitable peptide for reaction therewith (e.g., a
peptide lacking lysine and cysteine residues, but optionally
containing one or more other reactive nucleophilic amino
acids).
[0103] In some embodiments, provided herein are sulfo-SE peptides.
In some embodiments, peptides display a sulfo-SE group on the
N-terminus of the peptide. In some embodiments, peptide display a
sulfo-SE group on the C-terminus of the peptide. In some
embodiments, peptide display a sulfo-SE group on the side chain of
the peptide. In some embodiments, the peptide lacks lysine and
cysteine residues, but optionally containing one or more other
reactive nucleophilic amino acids. In some embodiments, sulfo-SE
peptides are universal reagents for conjugating peptides to targets
of interest (e.g., biomolecules).
[0104] In some embodiments, provided herein are methods for the
labeling a biomolecule with a peptide tag. In some embodiments, the
biomolecule comprises any suitable protein, polynucleotide,
polypeptide, biomolecular complex, natural product, synthetic
macromolecule, etc., that displays one or more amine groups (e.g.,
primary amine). In some embodiments, one or more amine groups are
displayed on the surface and/or a solvent accessible region of the
biomolecule. In some embodiments, the biomolecule comprises 1-100
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60,
70, 80, 90, 100, or ranges therebetween) amine (e.g., primary
amine) groups (e.g., accessible amine groups). In some embodiments,
a biomolecule for targeting with a sulfo-SE peptide is a protein,
enzyme, receptor, antibody, antibody fragment, polypeptide, toxin,
cytokine, polynucleotides, drugs, small molecules, ligand,
inhibitors, biomolecular complex (e.g., comprising one or more
components selected from protein(s), polynucleotides (e.g., DNA,
RNA), carbohydrates, lipids, small molecules, etc.), primary
metabolites, secondary metabolites, and natural products, exogenous
biomolecules, such as, pharmaceutical drugs, etc.
[0105] In some embodiments, provided herein are methods for the
labeling of biomolecules with reactive sulfo-SE peptides (FIG. 2).
In some embodiments, a biomolecule is combined with sulfo-SE
peptide (e.g., under appropriate reaction conditions (e.g., pH 4-9,
pH 7-8.5 (e.g., NaHCO.sub.3 solution (pH 8.5)) to produce a
peptide-labeled biomolecule through the reaction of the sulfo-SE
group with an amine (e.g., primary amine) on the biomolecule (e.g.,
on the surface of the biomolecule, a solvent accessible amine,
etc.).
[0106] Some embodiments herein find use in attaching bioluminescent
peptides or polypeptides, or components of bioluminescent complexes
(e.g., peptide capable of forming a complex with one or more other
peptide/polypeptide components) to biomolecules of interest. In
some embodiments, such labeled biomolecules are used in
bioluminescent-based assays and platforms. Some embodiments herein
utilize a NanoLuc-based bioluminescent platform or NanoLuc-based
bioluminescent complementation platform (See, e.g., WO/2014/151736
(Intl. App. No. PCT/US2014/026354) and U.S. Pro. App. No.
62/684,014; herein incorporated by reference in their
entireties).
[0107] NanoLuc.RTM. Binary Technology (NanoBiT) is a structural
complementation reporter designed for biomolecular interaction
studies. The NanoBiT.RTM. system is composed of two small
non-luminescent subunits, Large BiT (LgBiT; 18 kDa) and Small BiT
(SmBiT; 11 amino acid peptide) that have been optimized for
stability and minimal self-association. When two biomolecular
components labeled with these subunits come in close proximity, the
subunits come together to form an active enzyme and generate a
bright luminescent signal. The small subunit size of the NanoBiT
complementation partners minimizes interference with protein
functionality, and the bright signal allows sensitive detection.
HALOTAG and biotin/streptavidin systems have been the most common
methods to link the NanoBiT components to the targets of interests
(FIG. 3). However, both methods impose the significant complexity
for labeling a small peptide to the targets of interests and are
also limited by the large size of HALOTAG or streptavidin as the
linkage to form the optimal complementary active enzyme and
generate a bright luminescent signal.
[0108] Experiments conducted during development of embodiments
herein demonstrate synthesis of a stable (e.g., not autoreactive)
sulfo-SE/peptide conjugate (e.g., using the SmBiT peptide).
Experiments were also conducted to demonstrate peptide labeling of
biomolecules (e.g., antibodies, streptavidin) with a peptide
(sulfo-SE-SmBiT) at primary amines via a one-step SE protein
labeling protocol (FIG. 4).
[0109] Embodiments herein find use in attaching a reactive sulfo-SE
group to peptides or polypeptides with. The sulfo-SE group allows
the peptides or polypeptides to be conjugated to suitable
biomolecules that display an accessible amine group (e.g., primary
amine). In some embodiments, any peptides lacking lysine and
cysteine amino acids (lacking accessible lysine and cysteine amino
acids) is suitable for sulfo-SE labeling via the methods described
herein. In some embodiments, even peptides comprising one or more
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) reactive
nucleophilic amino acids (e.g., serine, threonine, arginine,
tyrosine, glutamic acid, aspartic acid) are amenable to
sulfo-SE-labeling by the methods herein.
[0110] In some embodiments, an SE-sulfo group on a peptide is
utilized to attach the peptide to a biomolecule or other analyte
for use in a bioluminescent complementation system. In some
embodiments, peptide and polypeptide components are non-luminescent
in the absence of complementation and/or complementation enhances
bioluminescence of a peptide or polypeptide component. In some
embodiments, target analyte binding agents (e.g., antibodies,
antibody fragments, etc.) are labeled with sulfo-SE-tagged peptide
components of the bioluminescent complexes described herein. For
example, embodiments of the present disclosure utilize sulfo-SE
labeling to incorporate NanoLuc-based technologies (e.g., NanoBit,
NanoTrip, NANO-GLO, NanoBRET, etc.) into target analyte detection
assays.
[0111] In some embodiments, provided herein are assays and
platforms using sulfo-SE peptides to incorporate bioluminescent
polypeptides and/or bioluminescent complexes (of peptide(s) and/or
polypeptide components) based on (e.g., structurally, functionally,
etc.) the luciferase of Oplophorus gracilirostris, the NanoLuc
luciferase (Promega Corporation; U.S. Pat. Nos. 8,557,970;
8,669,103; herein incorporated by reference in their entireties),
and/or the NanoBiT (U.S. Pat. No. 9,797,889; herein incorporated by
reference in its entirety) or NanoTrip (U.S. Prov. App. No.
62/684,014; herein incorporated by reference in its entirety). As
described below, in some embodiments, the assays, devices, and
systems herein incorporate commercially available NanoLuc-based
technologies (e.g., NanoLuc luciferase, NanoBRET, NanoBiT,
NanoTrip, Nano-Glo, etc.), but in other embodiments, various
combinations, variations, or derivations from the commercially
available NanoLuc-based technologies are employed.
[0112] PCT Appln. No. PCT/US2010/033,449, U.S. Pat. No. 8,557,970,
PCT Appln. No. PCT/2011/059,018, and U.S. Pat. No. 8,669,103 (each
of which is herein incorporated by reference in their entirety and
for all purposes) describe compositions and methods comprising
bioluminescent polypeptides; such polypeptides find use in
embodiments herein and can be used in conjunction with the assays
and methods described herein
[0113] PCT Appln. No. PCT/US14/26354 and U.S. Pat. No. 9,797,889
(each of which is herein incorporated by reference in their
entirety and for all purposes) describe compositions and methods
for the assembly of bioluminescent complexes (e.g., via the NanoBiT
system); such complexes, and the peptide and polypeptide components
thereof, find use in embodiments herein and can be used in
conjunction with the assays and methods described herein. In some
embodiments, sulfo-SE tagging of peptides is used to create
reactive peptides for linking to biomolecules of interests. In some
embodiments, any NanoBiT-based peptides or polypeptides are linked
(e.g., fused, chemically linked, etc.) to a binding element or
other component of the assays and systems described herein using
the methods described herein.
[0114] In some embodiments, provided herein are peptides having at
least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ
ID NO: 10 (SmBiT), but less than 100% (e.g., <99%, <98%,
<97%, <96%, <95%, <94%, <93%, <92%, <91%,
<90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID
NO: 5, and SEQ ID NO: 8. In some embodiments, such a peptide is
reacted with bis-sulfo-SE to produce a sulfo-SE peptide. In some
embodiments, provided herein are sulfo-SE peptides (N-terminal
labeled) comprising at least 60% (e.g., 06%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween)
sequence identity with SEQ ID NO: 10 (SmBiT), but less than 100%
(e.g., <99%, <98%, <97%, <96%, <95%, <94%,
<93%, <92%, <91%, <90%) sequence identity with SEQ ID
NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8, wherein the
peptide does not comprise lysine or cysteine. In some embodiments,
provided herein are sulfo-SE peptides comprising SEQ ID NO: 10
(SmBiT), wherein the peptide does not comprise lysine or cysteine.
In some embodiments, provided herein are methods of conjugating
such peptides to amines displayed on biomolecules. In some
embodiments, provided herein are biomolecules displaying such
peptides, following reaction of the sulfo-SE groups on the peptides
with amines on the biomolecules. assays and systems described
herein using the methods described herein.
[0115] In some embodiments, provided herein are polypeptides having
at least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with
SEQ ID NO: 9 (LgBiT), but less than 100% (e.g., <99%, <98%,
<97%, <96%, <95%, <94%, <93%, <92%, <91%,
<90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 5, and SEQ ID NO: 6. In some embodiments, such polypeptides for
bioluminescent complexes with complement peptides displayed on
biomolecules (following reaction of the sulfo-SE groups on the
peptides with amines on the biomolecules).
[0116] U.S. Prov. App. No. 62/684,014; herein incorporated by
reference in its entirety and for all purposes) describes
compositions and methods for the assembly of bioluminescent
complexes (e.g., via the NanoTrip system); such complexes, and the
peptides and polypeptide components thereof, find use in
embodiments herein and can be used in conjunction with the assays
and methods described herein. In some embodiments, sulfo-SE
attachment to peptides is used to create reactive peptides for
linking to biomolecules of interests. In some embodiments, any of
the aforementioned NanoTrip-based peptides or polypeptides are
linked (e.g., fused, chemically linked, etc.) to a binding element
or other component of the assays and systems described herein.
[0117] In some embodiments, provided herein are peptides having at
least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ
ID NO: 11 (HiBiT), but less than 100% (e.g., <99%, <98%,
<97%, <96%, <95%, <94%, <93%, <92%, <91%,
<90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID
NO: 5, and SEQ ID NO: 8. In some embodiments, such a peptide is
reacted with bis-sulfo-SE to produce a sulfo-SE peptide. In some
embodiments, provided herein are sulfo-SE peptides (N-terminal
labeled) comprising at least 60% (e.g., 06%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween)
sequence identity with SEQ ID NO: 11 (HiBiT), but less than 100%
(e.g., <99%, <98%, <97%, <96%, <95%, <94%,
<93%, <92%, <91%, <90%) sequence identity with SEQ ID
NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8, wherein the
peptide does not comprise lysine or cysteine. In some embodiments,
provided herein are sulfo-SE peptides comprising a lysine- and
cysteine-free variant of SEQ ID NO: 11 (HiBiT), wherein the peptide
does not comprise lysine or cysteine (e.g., any C or K residues are
conservatively substituted). In some embodiments, provided herein
are methods of conjugating such peptides to amines displayed on
biomolecules. In some embodiments, provided herein are biomolecules
displaying such peptides, following reaction of the sulfo-SE groups
on the peptides with amines on the biomolecules.
[0118] In some embodiments, provided herein are peptides having at
least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ
ID NO: 13 (SmTrip9), but less than 100% (e.g., <99%, <98%,
<97%, <96%, <95%, <94%, <93%, <92%, <91%,
<90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, and SEQ ID NO: 7. In some embodiments, such a peptide is
reacted with bis-sulfo-SE to produce a sulfo-SE peptide. In some
embodiments, provided herein are sulfo-SE peptides (N-terminal
labeled) comprising at least 60% (e.g., 06%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween)
sequence identity with SEQ ID NO: 13 (SmTrip9), but less than 100%
(e.g., <99%, <98%, <97%, <96%, <95%, <94%,
<93%, <92%, <91%, <90%) sequence identity with SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, wherein the
peptide does not comprise lysine or cysteine. In some embodiments,
provided herein are sulfo-SE peptides comprising SEQ ID NO: 13
(SmTrip9), wherein the peptide does not comprise lysine or cysteine
(e.g., any C or K residues are conservatively substituted). In some
embodiments, provided herein are methods of conjugating such
peptides to amines displayed on biomolecules. In some embodiments,
provided herein are biomolecules displaying such peptides,
following reaction of the sulfo-SE groups on the peptides with
amines on the biomolecules.
[0119] In some embodiments, provided herein are peptides having at
least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ
ID NO: 14 (.beta.9/.beta.10 dipeptide), but less than 100% (e.g.,
<99%, <98%, <97%, <96%, <95%, <94%, <93%,
<92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO:
8. In some embodiments, such a peptide is reacted with sulfo-NHS to
produce a sulfo-SE peptide. In some embodiments, provided herein
are sulfo-SE peptides (N-terminal labeled) comprising at least 60%
(e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
100%, or ranges therebetween) sequence identity with SEQ ID NO: 14
(.beta.9/.beta.10 dipeptide), but less than 100% (e.g., <99%,
<98%, <97%, <96%, <95%, <94%, <93%, <92%,
<91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8,
wherein the peptide does not comprise lysine or cysteine. In some
embodiments, provided herein are sulfo-SE peptides comprising SEQ
ID NO: 14 (.beta.9/.beta.10 dipeptide), wherein the peptide does
not comprise lysine or cysteine (e.g., any C or K residues are
conservatively substituted). In some embodiments, provided herein
are methods of conjugating such peptides to amines displayed on
biomolecules. In some embodiments, provided herein are biomolecules
displaying such peptides, following reaction of the sulfo-SE groups
on the peptides with amines on the biomolecules.
[0120] In some embodiments, provided herein are polypeptides having
at least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with
a lysine- and cysteine-free variant of SEQ ID NO: 12 (LgTrip), but
less than 100% (e.g., <99%, <98%, <97%, <96%, <95%,
<94%, <93%, <92%, <91%, <90%) sequence identity with
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID
NO: 9. In some embodiments, such polypeptides for bioluminescent
complexes with complement peptides displayed on biomolecules
(following reaction of the sulfo-SE groups on the peptides with
amines on the biomolecules).
[0121] In some embodiments, provided herein are peptides having at
least 60% (e.g., 06%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, or ranges therebetween) sequence identity with one
or more of SEQ ID NOS: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36. In some embodiments,
a peptide comprises less than 100% (e.g., <99%, <98%,
<97%, <96%, <95%, <94%, <93%, <92%, <91%,
<90%) sequence identity with any naturally occurring (e.g., SEQ
ID NOS: 1-4) or commercial (e.g., SEQ ID NOS: 5-8) sequences
provided herein. In some embodiments, such a peptide is reacted
with bis-sulfo-SE to produce a sulfo-SE peptide. In some
embodiments, provided herein are sulfo-SE peptides (N-terminal
labeled) comprising at least 60% (e.g., 06%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween)
sequence identity with one or more of SEQ ID NOS: 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35
or 36, but less than 100% (e.g., <99%, <98%, <97%,
<96%, <95%, <94%, <93%, <92%, <91%, <90%)
sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5,
and SEQ ID NO: 8, wherein the peptide does not comprise lysine or
cysteine. In some embodiments, provided herein are sulfo-SE
peptides comprising SEQ ID NOS: 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, wherein the
peptide does not comprise lysine or cysteine. In some embodiments,
provided herein are methods of conjugating such peptides to amines
displayed on biomolecules. In some embodiments, provided herein are
biomolecules displaying such peptides, following reaction of the
sulfo-SE groups on the peptides with amines on the
biomolecules.
[0122] As disclosed in PCT Appln. No. PCT/US13/74765 and U.S.
patent application Ser. No. 15/263,416 (herein incorporated by
reference in their entireties and for all purposes) describe
bioluminescence resonance energy transfer (BRET) system and methods
(e.g., incorporating NanoLuc-based technologies); such systems and
methods, and the bioluminescent polypeptide and
fluorophore-conjugated components thereof, find use in embodiments
herein and can be used in conjunction with the assays and methods
described herein
[0123] In some embodiments, any of the NanoLuc-based,
NanoBiT-based, and/or NanoTrip-based peptides (e.g., sulfo-SE
peptides, biomolecule conjugated peptides, etc.), polypeptide,
complexes, fusions, and conjugates may find use in BRET-based
applications with the assays, methods, devices, and systems
described herein. For example, an SmBiT peptide (or other NanoBiT-
or NanoTrip-based peptide described herein) is linked to a
biomolecule via the sulfo-SE technology described herein; a
bioluminescent complex is formed when the peptide is brought into
contact or proximity with one or more other components of the
system (e.g., linked to some target of interest, linked to a
binding agent, etc.); BRET is detected when the bioluminescent
complex is brought into proximity or contact with a fluorescent
molecule (e.g., linked to some target of interest, linked to a
binding agent, etc.). In some embodiments, the emission spectrum of
the NanoLuc-based, NanoBiT-based, and/or NanoTrip-based
polypeptide, peptide, or complex overlaps the excitation spectrum
of the fluorescent molecule (e.g., fluorophore). In some
embodiments, the fluorescent molecule (e.g., fluorophore) is an
energy acceptor. As used herein, the term "energy acceptor" refers
to any small molecule (e.g., chromophore), macromolecule (e.g.,
autofluorescent protein, phycobiliproteins, nanoparticle, surface,
etc.), or molecular complex that produces a readily detectable
signal in response to energy absorption (e.g., resonance energy
transfer). In certain embodiments, an energy acceptor is a
fluorophore or other detectable chromophore. Suitable fluorophores
include, but are not limited to: xanthene derivatives (e.g.,
fluorescein, rhodamine, Oregon green, eosin, Texas red, etc.),
cyanine derivatives (e.g., cyanine, indocarbocyanine,
oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene
derivatives (e.g., dansyl and prodan derivatives), oxadiazole
derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole,
benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue),
oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet,
oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine
orange, acridine yellow, etc.), arylmethine derivatives (e.g.,
auramine, crystal violet, malachite green, etc.), tetrapyrrole
derivatives (e.g., porphin, phthalocyanine, bilirubin, etc.), CF
dye (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen),
DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma
Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics),
SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA
BioMedicals), QUASAR, and CAL FLUOR dyes (Biosearch Technologies),
SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes)(Columbia
Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent
proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot
nanocrystals, etc. In some embodiments, a fluorophore is a
rhodamine analog (e.g., carboxy rhodamine analog), such as those
described in U.S. patent application Ser. No. 13/682,589, herein
incorporated by reference in its entirety.
[0124] As described above, the labeling technologies described
herein are not limited to use with bioluminescent peptides,
polypeptides, and complexes. Rather, the compositions, methods and
systems herein find use as a general system for the attachment of
peptides (e.g., peptide lacking a lysine and cysteine residue) to
biomolecules or materials. The peptide-conjugated biomolecules
generated herein find use in a variety of systems, reactions,
reagents, platforms, and assays.
[0125] A particular application for the technologies described
herein is linking peptides to biomolecules (e.g., antibodies,
antibody fragments, antigens, etc.) for use in ligand binding
and/or immunoassays. In some embodiments, the compositions and
methods herein find use in attaching peptide components of
immunoassays (e.g., antigens, components of bioluminescent
complexes, etc.) to other immunoassay components (e.g., antibodies,
antibody fragments, antigens, etc.). Embodiments herein find use in
preparing reagents for, and/or are involved in the steps of,
various immunoassays, such as competitive immunoassays, direct
immunoassays, indirect immunoassays, enzyme linked immunosorbent
assays (ELISA), sandwich immunoassay, combination immunoassays
(e.g., See U.S. application Ser. No. 15/589,557; herein
incorporated by reference in its entirety), etc. In various
embodiments, a sulfo-SE peptide tag (e.g., .beta.9-like (e.g.,
SmTrip9) and .beta.10-like (e.g., SmTrip10) peptides;
.beta.9/.beta.10-like dipeptides; etc.) is tethered/fused to a
primary or secondary antibody (e.g., at a primary amine) to provide
a method of detection for a particular analyte. As another example,
a sulfo-SE peptide tag is tethered/fused to an antibody-binding
protein (e.g., protein A or protein G) and used to detect a
specific antibody bound to a particular analyte (e.g., wherein the
analyte is bound to the complementary peptide tag). As another
example, a sulfo-SE peptide tag is tethered/fused to streptavidin
and used to detect a specific biotinylated antibody bound to a
particular analyte (e.g., wherein the analyte is bound to the
complementary peptide tag). As yet another example, sulfo-SE
peptide tags are tethered/fused to primary and secondary
antibodies, where the primary antibody recognizes a particular
analyte, and the secondary antibody recognizes the primary
antibody. As still another example, a sulfo-SE peptide tag is
tethered/fused to an analyte and used in a competitive sandwich
ELISA format. A sulfo-SE peptide tag is tethered/fused conjugated
to an analyte may also be used to detect antibodies capable of
binding the analyte.
[0126] Various embodiments herein find use in small molecule or
other biomolecule detection via immunoassay. Exemplary embodiments
comprise the use of a small molecule or other biomolecule directly
(e.g., identical or similar to the target small molecule) labeled
with a first sulfo-SE peptide tag (e.g., a first peptide component
of a bioluminescent complex) described herein and a binding moiety
for the target small molecule or biomolecule is fused or linked to
a second sulfo-SE peptide tag (e.g., a second peptide component of
a bioluminescent complex) described herein. In the presence of
detection reagents (e.g., polypeptide component of the
bioluminescent complex and substrate (e.g., coelenterazine or
coelenterazine analog)), a bioluminescent signal is produced by the
system. When the system is exposed to a sample (e.g., biological
sample, environmental sample, etc.), the bioluminescent signal will
be reduced if the small molecule or biomolecule target is present
in the sample (the labeled small molecule or biomolecule will be
competed out of the complex allowing, in some cases, quantitation
of the small molecule or biomolecule target). Alternative
configurations for such assays are also within the scope herein
(e.g., a biomolecular complex comprising a single sulfo-SE
component).
[0127] In some embodiments, the analyte for an immunoassay is a
toxin (e.g., mycotoxin, etc.), metabolite (e.g., amino acid,
glucose molecule, fatty acid, nucleotide, cholesterol, steroid,
etc.), vitamin (e.g., vitamin A, vitamin B1, vitamin B2, Vitamin
B3, vitamin B5, vitamin B7, vitamin B9, vitamin B12, vitamin C,
vitamin D, vitamin E, vitamin H or vitamin K, etc.), coenzyme or
cofactor (e.g., coenzyme A, coenzyme B, coenzyme M, coenzyme Q,
cytidine triphosphate, acetyl coenzyme A, reduced nicotinamide
adenine dinucleotide (NADH), nicotinamide adenine (NAD+),
nucleotide adenosine monophosphate, nucleotide adenosine
triphosphate, glutathione, heme, lipoamide, molybdopterin,
3'-phosphoadenosine-5'-phosphosulfate, pyrroloquinoline quinone,
tetrahydrobiopterin, etc.), biomarker or antigen (e.g.,
erythropoietin (EPO), ferritin, folic acid, hemoglobin, alkaline
phosphatase, transferrin, apolipoprotein E, CK, CKMB, parathyroid
hormone, insulin, cholesteryl ester transfer protein (CETP),
cytokines, cytochrome c, apolipoprotein AI, apolipoprotein AII,
apolipoprotein BI, apolipoprotein B-100, apolipoprotein B48,
apolipoprotein CII, apolipoprotein CIII, apolipoprotein E,
triglycerides, HD cholesterol, LDL cholesterol, lecithin
cholesterol acyltransferase, paraoxonase, alanine aminotransferase
(ALT), asparate transferase (AST), CEA, HER-2, bladder tumor
antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA 19.9, CA
15.3, leptin, prolactin, osteopontin, CD 98, fascin, troponin I,
CD20, HER2, CD33, EGFR, VEGFA, etc.), drug (cannabinoid (e.g.,
tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN),
etc.), opioid (e.g., heroin, opium, fentanyl, etc.), stimulant
(e.g., cocaine, amphetamine, methamphetamine, etc.), club drug
(e.g., MDMA, flunitrazepam, gama-hydroxybutyrate, etc.),
dissociative drug (e.g., ketamine, phencyclidine, salvia,
dextromethorphan, etc.), hallucinogens (e.g., LSD, mescaline,
psilocybin, etc.), etc.), explosive (e.g., 2,4,6-trinitrotoluene
(TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX),
pentaerythritol tetranitrate (PETN), etc.), toxic chemical (e.g.,
tabun (GA), sarin (GB), soman (GD), cyclosarin (GF),
2-(dimethylamino)ethyl N, N-dimethylphosphoramidofluroidate (GV),
VE, VG, VM, VP, VR, VS, or VX nerve agent), etc.
[0128] In some embodiments, the sulfo-SE peptide is attached to the
analyte. In some embodiments, the sulfo-SE peptide is attached to
an antibody or antibody fragment for the analyte. In some
embodiments, the sulfo-SE peptide is attached to streptavidin. In
some embodiments, the sulfo-SE peptide is attached to HALOTAG. In
some embodiments, sulfo-SE peptide is attached to a solid surface.
Various immunoassays or other assays can be carried out using such
reagents and are within the scope herein.
[0129] The sulfo-SE peptides, and biomolecules labeled with the
sulfo-SE peptides herein (and methods of preparation thereof), find
use in a wide variety of applications and formats. The following
are non-exhaustive exemplary examples of methods and formats
utilizing the systems described herein: [0130] Intracellular, two
protein systems for dynamic protein-protein interaction analysis
with peptide-labeled proteins expressed as fusions via traditional
transfection or endogenously tagged proteins via CRISPR; [0131]
Intracellular, three protein systems for dynamic protein-protein
interaction analysis with peptide-labeled proteins expressed as
fusions via traditional transfection or as endogenously-tagged
proteins generated via CRISPR; [0132] Target specific assays for
analyte measurement by gain of signal (e.g. diagnostic test,
non-cellular, etc.); [0133] Target specific competition assays for
analyte measurement through loss of signal (e.g. diagnostic test,
noncellular, etc.); [0134] Homogeneous assays using peptide
tag-labelled recognition elements for detection/quantification of a
single analyte or multiple analytes; [0135] Detection of analyte(s)
in liquid/solution phase or solid phase. [0136] Surface-based
assays (e.g., plate-based (e.g., microtiter plate), paper-based
(e.g., Whatman protein saver 903 cards), plastic-based, swab-based,
cuvette-based, membrane-based (e.g., PVDF, nitrocellulose, etc.),
etc.; [0137] Lateral flow and other capillary driven based methods;
[0138] Plate-based for solution phase assay (e.g., performed in a
multiplexed dot blot/spot array assay format); [0139] Aerosol-based
detection; [0140] Isothermal amplification of nucleic acids; [0141]
Rapid cycling PCR detection of nucleic acids; [0142] Detection of
protein-protein interaction; [0143] Detection of native proteins in
heterogeneous solutions; [0144] Peptide-tagged complimentary
recognition elements hybridize to a nucleic acid target sequence in
tandem; [0145] FISH-like applications utilizing bioluminescence or
BRET for detection/quantification; [0146] Detection of nucleic
acids (e.g., single stranded and/or double stranded DNA and/or
RNA); [0147] Lab-on-chip and/or microfluidics applications; [0148]
Heterogeneous assays such as immunoassays (e.g., PCR amplification
combined with homogeneous immunoassay analysis); [0149] Etc.
[0150] Embodiments herein are not limited by the end uses of the
sulfo-SE peptides and biomolecules labeled therewith.
[0151] Although embodiments herein are described as useful for
linking a peptide (e.g., labeled with a sulfo-SE group) to various
biomolecules, embodiments herein are not so limited. In some
embodiments, sulfo-SE peptides are reacted with other molecules,
molecular entities, materials, etc. in order to link and/or
immobilize the peptide thereto. For example, a sulfo-SE peptide may
be attached to a solid surface (e.g., beads (e.g., magnetic beads),
chips, tubes, plates, particles, membranes, paper, etc.) that
displays an amine group using the chemistry and reagents described
herein.
EXPERIMENTAL
[0152] Experiments were conducted during development of embodiments
herein to demonstrate the synthesis of sulfo-SE SmBiT peptides,
conjugation of sulfoSE and peptide with different linkers, and
conjugation of sulfo-SE-SmBiT with other moieties such as
fluorophores, to demonstrate the utility of peptide-Sulfo-SE
labeling of various antigens and the usefulness of such labeled
antigens in various assay formats with various target antigens, the
utility of peptide direct labeling, etc. These experiments
demonstrate a portion of the breadth of the useful applications of
the present technology but should not be viewed as limiting of the
scope herein.
Example 1
##STR00003##
[0154] Small Bit peptide (VTGYRLFEEIL, 6 mg, 0.0045 mmol) was
dissolved in a minimum amount of DMF and then added to a solution
of BS3 (bis(sulfosuccinimidyl)suberate) (13 mg, 0.013 mmol) in
phosphate buffer (0.5 M, pH=7.4). The reaction mixture was stirred
for 1 h and directly purified by preparative HPLC. Calculated:
m/z=1672.79 [M+H].sup.+; measured (ESI): m/z=1672.75.
Example 2
##STR00004##
[0156] 7962 was synthesized by the same method as 7649. Calculated:
m/z=1707.85 [M+H].sup.+; measured (ESI): m/z=1707.75.
Example 3
##STR00005##
[0158] 8084 was synthesized by the same method as 7649. Calculated:
m/z=1006.99 [M+2H].sup.2+; measured (ESI): m/z=1006.32
[M+2H].sup.2+.
Example 4
##STR00006##
[0160] 3,3'-((oxybis(ethane-2,1-diyl))bis(oxy))dipropionic acid (55
mg, 0.22 mmol) was dissolved in anhydrous DMF, and then
diisopropylethylamine (120 mg, 0.88 mmol) and HATU (176 mg, 0.45
mmol) added. The mixture was stirred for five minutes. Meanwhile,
N-hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid (90 mg, 0.46 mmol)
was dissolve in 5 ml DMSO, and then added to the previous solution
dropwise. The mixture was stirred for another hour until LC-MS
shows disappearance of acid. The solution was directly used in the
next step. Calculated: m/z=603.05 [M.sup.-]; measured (ESI):
m/z=603.04 [M.sup.-].
##STR00007##
SmTrip9 (693) Peptide Trip521 (GRMLFRVTINSWR, 27 mg, 0.045 mmol)
was dissolved in DMF. The solution was then added to the previous
PEG3 bis Sulfo-SE solution. The mixture was then stirred for
another hour and directly purified by preparative HPLC. Calculated:
m/z=1022.98 [M+2H].sup.2+; measured (ESI): m/z=1023.09
[M+2H].sup.2+.
Example 5
##STR00008##
[0162] 8084 was synthesized by the same method as 8134. Calculated:
m/z=892.93 [M+2H].sup.2+; measured (ESI): m/z=893.61
[M+2H].sup.2+.
Example 6
##STR00009##
[0164] 8160 was synthesized by the same method as 8134. Calculated:
m/z=1016.51 [M+2H].sup.2+; measured (ESI): m/z=1016.92
[M+2H].sup.2+.
Example 7
##STR00010##
[0166] 5-TAMRA (50 mg, 0.116 mmol) was dissolved in DMF.
Diisopropylethylamine (45 mg, 0.128 mmol) was added followed by
TSTU (38 mg, 0.128 mmol). The mixture was stirred for 20 min, and
1-(2-aminoethyl)-1H-pyrrole-2,5-dione (18 mg, 0.128 mmol) was
added. The resulting reaction mixture was stirred for another hour
and directly purified by preparative HPLC. Calculated: m/z=553.20
[M+H].sup.+; measured (ESI): m/z=553.40.
##STR00011##
[0167] TAMRA-Maleimide (8 mg, 0.014 mmol) was dissolved in DMF. A
solution of SmTrip9 (938) (GRMLFRVTINSWRC, 25 mg, 0.014 mmol) in
PBS buffer (pH 7.4, 200 mM) was added. The reaction mixture was
stirred for two hours and directly purified by preparative HPLC.
Calculated: m/z=1146.05 [M+2H].sup.2+; measured (ESI): m/z=1146.33
[M+2H].sup.2+.
##STR00012##
SmTrip9 (938)-TAMRA (8.5 mg, 0.0038 mmol) was dissolved in DMF. The
solution was then added to PEGS bis Sulfo-SE prepared as shown in
Example 2. The reaction mixture was stirred for two hours and
directly purified by preparative HPLC. Calculated: m/z=901.05
[M+3H].sup.3+; measured (ESI): m/z=901.20 [M+3H].sup.3+.
Example 8
##STR00013##
[0169] 8135 was synthesized by the same method as 8136. Calculated:
m/z=814.03 [M+3H].sup.3+; measured (ESI): m/z=814.40
[M+3H].sup.3+.
Example 9
##STR00014##
[0171] 8161 was synthesized by the same method as 8136. Calculated:
m/z=896.61 [M+3H].sup.3+; measured (ESI): m/z=897.11
[M+3H].sup.3+.
Example 10
Sulfo-SE-SmBiT Conjugation to an Exemplary Target Analyte
[0172] Sulfo-SE-SmBiT (7649) was dissolved in DMF at a
concentration of 6 mM. A 20.times. molar ratio amount was added to
a 1 mg/ml solution of goat anti-mouse IgG in pH 8.2 borate buffer.
The reaction was mixed at room temperature for 1 hour. Unreacted
sulfo-SE-SmBiT was removed by desalting column.
Example 11
Direct Immunoassay: Detection of Mouse IgG
[0173] A solution of anti-mouse IgG-sulfo-SE-SmBiT conjugate was
prepared in PBS containing 10% of SuperBlock blocking agent. This
was added to an equal volume of equal concentration anti-mouse
IgG-HT-LgBiT conjugate also in PBS containing 10% of SuperBlock
blocking agent. The mixture was dispensed into wells of a white,
non-binding 96-well microtiter plate. Mouse IgG was serially
diluted in PBS containing 10% of SuperBlock blocking agent and
added to the wells of the plate in a volume equal to that of the
anti-mouse IgG-sulfo-SE-SmBiT conjugate. The plate was placed on a
platform plate shaker for 30 minutes. LCS Nano-Glo.RTM. detection
reagent was added, and luminescence read. For purposes of
comparison, this assay also included anti-mouse-HT-SmBit in place
of the anti-mouse IgG-sulfo-SE-SmBiT conjugate.
Example 12
Indirect Immunoassay: Detection of IFN.gamma.
[0174] IFN.gamma. was detected using indirect immunoassay. Paired
antibodies against IFN.gamma. were used. One antibody was
monoclonal antibody (mAb) while the other was biotinylated
polyclonal antibody (pAb-Biotin). Paired antibodies were mixed with
anti-Mouse IgG-LgBiT and Sav-SmBiT (HALOTAG OR Sulfo-SE-SmBiT) to
make a detection reagent. Detection reagent was added to IFN.gamma.
and incubated for 30-60 min. NanoLuc.RTM. substrate was added, and
the plate read on a Glomax.RTM. luminometer.
Example 13
Competition Immunoassay: Fumonisin
[0175] A fumonisin "tracer" was prepared by combining Sav-SmBiT
(HALOTAG OR sulfo-SE-SmBiT) and biotinylated fumonisin to yield a
fumonisin-SAv-SmBiT conjugate. This "tracer" was diluted to 1 ug/ml
in PBS containing 10% SuperBlock blocking agent and added to a
white, non-binding 96-well microtiter plate. Unlabeled fumonisin
was serially diluted in PBS containing 10% SuperBlock blocking
agent, and an equal volume was added to the wells of the plate.
Anti-fumonisin-LgBiT was prepared in PBS containing 10% SuperBlock
blocking agent, and an equal volume was added to the wells of the
plate. The plate was placed on a platform plate shaker for 30
minutes. LCS Nano-Glo.RTM. detection reagent was added, and
luminescence read.
Example 14
FcRn Binding Assay
[0176] FcRn-Avitag and Sav-SmBiT (HALOTAG or Sulfo-SE-SmBiT) were
mixed to make FcRn-SmBiT reagent. 25 ul of human IgG1-LgBiT tracer,
25 ul of human IgG sample, and 50 ul of FcRn-SmBiT were incubated
together for 30 min at room temperature. Reagents and samples were
diluted in pH6.0 PBS containing 10% superblock. NanoLuc.RTM.
substrate diluted in pH 6.0 dilution buffer was added, and the
plate read in a Glomax.RTM. luminometer.
Example 15
Bioluminescent Quantitation of Human Recombinant IL-6 Using
NanoTrip Directly Labeled Paired Antibodies with Sulfo-SE Peptide
Moieties without Built-In Linkers
[0177] Experiments were conducted during development of embodiments
herein to demonstrate the use of paired monoclonal antibodies that
have been directly chemically conjugated with NanoTrip peptides to
quantitation human IL-6. This model system consists of two
monoclonal mouse antibodies that recognize IL-6 at different
epitopes. Sulfo-SE-SmTrip9(824) (SEQ ID NO: 25) was chemically
conjugated to one of the antibodies, and Sulfo-SE-SmTrip10(691)
(SEQ ID NO: 23) was chemically conjugated to the other antibody. In
the presence of IL-6, the two antibodies bind to the IL-6 thus
bringing the two tags in close proximity. Addition of LgTrip (3546)
(SEQ ID NO: 12) completes the complementation, and a luminescent
signal is generated.
[0178] Sulfo-SE-SmTrip9(824) (SEQ ID NO: 25) and
Sulfo-SE-SmTrip10(691) (SEQ ID NO: 23) were created. Anti-IL-6
mouse monoclonal antibody clone 505E9A12A3 (Thermo) was labeled
with the Sulfo-SE-SmTrip10(691) (SEQ ID NO: 23) and anti-IL-6 mouse
monoclonal antibody clone 5IL6 (Thermo) was labeled with the
Sulfo-SE-SmTrip9(824)-R (SEQ ID NO: 25). The unlabeled antibodies
were prepped by first doing a buffer exchange into 10 mM
NaHCO.sub.3 (pH 8.5) performed 2.times. using Zeba columns.
Antibodies were then directly labeled with a 20-fold excess of the
respective reactive peptide and allowed to incubate at room
temperature while shaking at 1000 rpm for 2 hours. A buffer
exchange was done 2.times. using Zeba columns to remove free
linker.
[0179] A 2.times. stock of recombinant human IL-6 was generated in
assay buffer, serially diluted 1:2 to create a dose response, and
50 ul/well added to a non-binding surface treated, 96 well
solid-white plate (Costar 3600). A 2.times. master mix of the
purified LgTrip (3546) (SEQ ID NO: 12) (final concentration 1
uM)+Sulfo-SE-SmTrip9(824) (SEQ ID NO: 25) labeled 5IL6 clone (final
10 ng/ml)+Sulfo-SE-SmTrip10 (691) SEQ ID NO: 23) labeled 505E clone
(final 10 ng/ml) was created in assay buffer, and 50 ul/well added.
Plates were allowed to incubate for 90 minutes prior to addition of
a 5.times. stock of Nano-Glo.RTM. Live Cell Substrate in assay
buffer, 25 ul/well added to the plate for a final concentration of
10 uM, and luminescence measured using a GloMax.RTM. Discover.
Assay buffer consisted of Blocker BSA (10%) (Thermo) diluted in PBS
(pH 7.0) to a final of 0.01% BSA in PBS. Samples were tested in
triplicate.
[0180] Results are depicted in FIG. 10 as raw RLU (10A) or as
calculated signal to noise (10B) where signal to noise=(raw
RLU-background RLU)/standard deviation of background.
Example 16
Bioluminescent Quantitation of Human Recombinant IL-6 Using
NanoTrip Directly Labeled Paired Antibodies with Sulfo-SE Peptide
Moieties with Built-In Linkers
[0181] Experiments were conducted during development of embodiments
herein to demonstrate the use of paired monoclonal antibodies that
have been directly chemically conjugated with NanoTrip peptides to
quantitation human IL-6. This model system consists of two
monoclonal mouse antibodies that recognize IL-6 at different
epitopes. Sulfo-SE-PEG3-SmTrip9(693) (SEQ ID NO: 16) or
Sulfo-SE-PEG3-SmTrip9(895) (SEQ ID NO: 18) was chemically
conjugated to one of the antibodies, and
Sulfo-SE-PEG3-SmTrip10(691)(SEQ ID NO: 23 was chemically conjugated
to the other antibody. In the presence of IL-6, the two antibodies
bind to the IL-6 thus bringing the two tags in close proximity.
Addition of LgTrip (3546) (SEQ ID NO: 12) completes the
complementation, and a luminescent signal is generated.
[0182] Sulfo-SE-PEG3-SmTrip9(693) (SEQ ID NO: 16),
Sulfo-SE-PEG3-SmTrip9(895) (SEQ ID NO: 18) and
Sulfo-SE-SmTrip10(691) (SEQ ID NO: 23) are created. Anti-IL-6 mouse
monoclonal antibody clone 505E9A12A3 (Thermo) is labeled with the
Sulfo-SE-PEG3-SmTrip10(691) (SEQ ID NO: 23) and anti-IL-6 mouse
monoclonal antibody clone 5IL6 (Thermo) is labeled with the
Sulfo-SE-SmTrip9(824) (SEQ ID NO: 25) or the
Sulfo-SE-PEG3-SmTrip9(895) (SEQ ID NO: 18). The unlabeled
antibodies are prepped by first doing a buffer exchange into 10 mM
NaHCO.sub.3 (pH 8.5) performed 2.times. using Zeba columns.
Antibodies are then directly labeled with a fixed concentration of
200 uM of the respective reactive peptide and allowed to incubate
at room temperature while shaking at 1000 rpm for 2 hours. A buffer
exchange is done 2.times. using Zeba columns to remove free linker.
Samples are run on an SDS PAGE total protein gel and analyzed by
bright field imaging to indicate antibody labeling and determine
how much excess unreacted peptide remains as shown in FIG. 14A.
[0183] A 2.times. stock of recombinant human IL-6 was generated in
assay buffer, serially diluted 1:2 to create a dose response, and
50 ul/well added to a non-binding surface treated, 96 well
solid-white plate (Costar 3600). A 2.times. master mix of the
purified LgTrip (3546) (SEQ ID NO: 12) (final concentration 1
uM)+Sulfo-SE-PEG3-SmTrip9(693) (SEQ ID NO: 16) labeled 5IL6 clone
(final 10 ng/ml) or Sulfo-SE-PEG3-SmTrip9(895) (SEQ ID NO: 18)
labeled 5IL6 clone (final 10 ng/ml)+Sulfo-SEPEG3-SmTrip10 (691)
(SEQ ID NO: 23) labeled 505E clone (final 10 ng/ml) was created in
assay buffer, and 50 ul/well added. Plates were allowed to incubate
for 90 minutes prior to addition of a 5.times. stock of
Nano-Glo.RTM. Live Cell Substrate in assay buffer, 25 ul/well added
to the plate for a final concentration of 10 uM, and luminescence
measured using a GloMax.RTM. Discover. Assay buffer consisted of
Blocker BSA (10%) (Thermo) diluted in PBS (pH 7.0) to a final of
0.01% BSA in PBS. Samples were tested in triplicate.
[0184] Results are depicted in FIG. 11 as raw RLU (11A) or as
calculated signal to noise (11B) where signal to noise=(raw
RLU-background RLU)/standard deviation of background.
Example 17
NanoBRET Quantitation of Human Recombinant IL-6 Using NanoTrip
Directly Labeled
[0185] Paired Antibodies with Sulfo-SE Peptide Moieties with
Built-in Linkers Experiments were conducted during development of
embodiments herein to demonstrate the use of paired monoclonal
antibodies that have been directly chemically conjugated with
NanoTrip peptides to quantitation human IL-6. This model system
consists of two monoclonal mouse antibodies that recognize IL-6 at
different epitopes. Sulfo-SE-PEG3-SmTrip9(938)-TAMRA (SEQ ID NO:
38) was chemically conjugated to one of the antibodies, and
Sulfo-SE-PEG3-SmTrip10 (937)-TAMRA (SEQ ID NO: 37) was chemically
conjugated to the other antibody. In the presence of IL-6, the two
antibodies bind to the IL-6 thus bringing the two tags in close
proximity. Addition of LgTrip (3546) (SEQ ID NO: 12) completes the
complementation, and a luminescent signal is generated which in
turn excites the TAMRA acceptor fluorophore allowing it to emit
light at 580 nm. The donor luminescence signal and acceptor
fluorophore signal are analyzed using the NanoBRET ratio of
acceptor signal/donor signal is calculated.
[0186] Sulfo-SE-PEG3-SmTrip9(938)-TAMRA (SEQ ID NO: 38) and
Sulfo-SE-PEG3-SmTrip10 (937)-TAMRA (SEQ ID NO: 37) were created.
Anti-IL-6 mouse monoclonal antibody clone 505E9A12A3 (Thermo) was
labeled with the Sulfo-SE-PEG3-SmTrip10-TAMRA (SEQ ID NO: 11) and
anti-IL-6 mouse monoclonal antibody clone 5IL6 (Thermo) was labeled
with the Sulfo-SE-PEG3-SmTrip9(521)-TAMRA (SEQ ID NO: 26) or the
Sulfo-SE-PEG3-SmTrip9(895) (SEQ ID NO: 18). The unlabeled
antibodies were prepped by first doing a buffer exchange into 10 mM
NaHCO.sub.3 (pH 8.5) performed 2.times. using Zeba columns.
Antibodies were then directly labeled with a fixed concentration of
200 uM of the respective reactive peptide and allowed to incubate
at room temperature while shaking at 1000 rpm for 2 hours. A buffer
exchange was done 2.times. using Zeba columns to remove free
linker. Samples were run on an SDS PAGE total protein gel and
analyzed by bright field imaging (FIG. 14A) or fluorescent imaging
(FIG. 14B) to indicate antibody labeling and to determine how much
excess unreacted peptide remains.
[0187] A 2.times. stock of recombinant human IL-6 was generated in
assay buffer and 50 ul/well added to a non-binding surface treated,
96 well solid-white plate (Costar 3600) (Final concentration 20
ng/ml). A 2.times. master mix of the purified LgTrip (3546) (SEQ ID
NO: 12) (final concentration 1 uM)+Sulfo-SE-PEG3-SmTrip9(938)-TAMRA
(SEQ ID NO: 38) labeled 5IL6 clone (final 10 ng/ml) 5IL6 clone
(final 10 ng/ml)+Sulfo-SE-PEG3-SmTrip10 (937)-TAMRA (SEQ ID NO: 37)
labeled 505E clone (final 10 ng/ml) was created in assay buffer,
and 50 ul/well added. A5.times. stock of Nano-Glo.RTM. Live Cell
Substrate in assay buffer, 25 ul/well added to the plate for a
final concentration of 10 uM, and BRET was measured using a
GloMax.RTM. Discover in real time. Assay buffer consisted of
Blocker BSA (10%) (Thermo) diluted in PBS (pH 7.0) to a final of
0.01% BSA in PBS. Samples were tested in triplicate. Results are
depicted in FIG. 13 as raw RLU over time.
[0188] It is understood that the foregoing detailed description and
accompanying examples are merely illustrative and are not to be
taken as limitations upon the scope of the disclosure, which is
defined solely by the appended claims and their equivalents.
[0189] Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such
changes and modifications, including without limitation those
relating to the chemical structures, substituents, derivatives,
intermediates, syntheses, compositions, formulations, or methods of
use of the disclosure, may be made without departing from the
spirit and scope thereof.
TABLE-US-00002 SEQUENCES WT OgLuc (SEQ ID NO: 1)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSG
ENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTL
VIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPD
GSLLFRVTINGVTGWRLCENILA WT OgLuc Lg (SEQ ID NO: 2)
MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSG
ENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTL
VIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPD WT OgLuc .beta.9
(SEQ ID NO: 3) GSLLFRVTIN WT OgLuc .beta.10 (SEQ ID NO: 4)
GVTGWRLCENILA NanoLuc (SEQ ID NO: 5)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS
GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP
DGSLLFRVTINGVTGWRLCERILA NanoLuc Lg (SEQ ID NO: 6)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS
GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP D NanoLuc 119
(SEQ ID NO: 7) GSLLFRVTINV NanoLuc 1310 (SEQ ID NO: 8)
GVTGWRLCERILA LgBiT (SEQ ID NO: 9)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS
GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP DGSLLFRVTIN SmBiT
(SEQ ID NO: 10) VTGYRLFEEIL HiBiT (pep86) (SEQ ID NO: 11)
VSGWRLFKKIS LgTrip (3546) (SEQ ID NO: 12)
MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVT
PIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHH
FKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGN KIIDERLITPD
SmTrip9 (SEQ ID NO: 13) GSMLFRVTINS .beta.9/.beta.10 dipeptide (SEQ
ID NO: 14) GSMLFRVTINSVSGWRLFKKIS Pep521 (SEQ ID NO: 15)
GKMLFRVTINSWK Pep693 (SEQ ID NO: 16) GRMLFRVTINSWR Pep840 (SEQ ID
NO: 17) GKLLFVVVIEKYK Pep895 (SEQ ID NO: 18) GRLLFVVVIERYR Pep 760
(SEQ ID NO: 19) KKMLFRVTIQKWK Pep929 (SEQ ID NO: 20) RRMLFRVTIQRWR
VS-HiBiT (Pep289) (SEQ ID NO: 21) VSVSGWRLFKKIS Pep692 (SEQ ID NO:
22) VSVSGWRLFRRIS Pep691 (SEQ ID NO: 23) VSGWRLFRRIS Pep 759 (SEQ
ID NO: 24) DKLLFTVTIEKYK Pep824 (SEQ ID NO: 25) DRLLFTVTIERYR
Pep521-C (SEQ ID NO: 26) GKMLFRVTINSWKC Pep693-C (SEQ ID NO: 27)
GRMLFRVTINSWRC Pep840-C (SEQ ID NO: 28) GKLLFTVTIEKYKC Pep895-C
(SEQ ID NO: 29) GRLLFTVTIERYRC Pep 760-C (SEQ ID NO: 30)
KKMLFRVTIQKWKC Pep929-C (SEQ ID NO: 31) RRMLFRVTIQRWRC VS-HiBiT-C
(Pep289) (SEQ ID NO: 32) VSVSGWRLFKKISC Pep692-C (SEQ ID NO: 33)
VSVSGWRLFRRISC Pep691-C (SEQ ID NO: 34) VSGWRLFRRISC Pep 759-C (SEQ
ID NO: 35) DKLLFTVTIEKYKC Pep824-C (SEQ ID NO: 36) DRLLFTVTIERYRC
Pep937 (SEQ ID NO: 37) VSGWRLFRRISC Pep938 (SEQ ID NO: 38)
GRMLFRVTINSWRC Pep939 (SEQ ID NO: 39) GRLLFTVTIERYRC
Sequence CWU 1
1
391170PRTArtificial sequenceSynthetic 1Met Phe Thr Leu Ala Asp Phe
Val Gly Asp Trp Gln Gln Thr Ala Gly1 5 10 15Tyr Asn Gln Asp Gln Val
Leu Glu Gln Gly Gly Leu Ser Ser Leu Phe 20 25 30Gln Ala Leu Gly Val
Ser Val Thr Pro Ile Gln Lys Val Val Leu Ser 35 40 45Gly Glu Asn Gly
Leu Lys Ala Asp Ile His Val Ile Ile Pro Tyr Glu 50 55 60Gly Leu Ser
Gly Phe Gln Met Gly Leu Ile Glu Met Ile Phe Lys Val65 70 75 80Val
Tyr Pro Val Asp Asp His His Phe Lys Ile Ile Leu His Tyr Gly 85 90
95Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly
100 105 110Arg Pro Tyr Pro Gly Ile Ala Val Phe Asp Gly Lys Gln Ile
Thr Val 115 120 125Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Tyr Asp
Glu Arg Leu Ile 130 135 140Asn Pro Asp Gly Ser Leu Leu Phe Arg Val
Thr Ile Asn Gly Val Thr145 150 155 160Gly Trp Arg Leu Cys Glu Asn
Ile Leu Ala 165 1702147PRTArtificial sequenceSynthetic 2Met Phe Thr
Leu Ala Asp Phe Val Gly Asp Trp Gln Gln Thr Ala Gly1 5 10 15Tyr Asn
Gln Asp Gln Val Leu Glu Gln Gly Gly Leu Ser Ser Leu Phe 20 25 30Gln
Ala Leu Gly Val Ser Val Thr Pro Ile Gln Lys Val Val Leu Ser 35 40
45Gly Glu Asn Gly Leu Lys Ala Asp Ile His Val Ile Ile Pro Tyr Glu
50 55 60Gly Leu Ser Gly Phe Gln Met Gly Leu Ile Glu Met Ile Phe Lys
Val65 70 75 80Val Tyr Pro Val Asp Asp His His Phe Lys Ile Ile Leu
His Tyr Gly 85 90 95Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile
Asp Tyr Phe Gly 100 105 110Arg Pro Tyr Pro Gly Ile Ala Val Phe Asp
Gly Lys Gln Ile Thr Val 115 120 125Thr Gly Thr Leu Trp Asn Gly Asn
Lys Ile Tyr Asp Glu Arg Leu Ile 130 135 140Asn Pro
Asp145310PRTArtificial sequenceSynthetic 3Gly Ser Leu Leu Phe Arg
Val Thr Ile Asn1 5 10413PRTArtificial sequenceSynthetic 4Gly Val
Thr Gly Trp Arg Leu Cys Glu Asn Ile Leu Ala1 5 105171PRTArtificial
sequenceSynthetic 5Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp
Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly
Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro
Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp
Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met
Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp
Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile
Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro
Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val
Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135
140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly
Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 165
1706148PRTArtificial sequenceSynthetic 6Met Val Phe Thr Leu Glu Asp
Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln
Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly
Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn
Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu
Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val
Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90
95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe
100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys
Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile
Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp145711PRTArtificial
sequenceSynthetic 7Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Val1 5
10813PRTArtificial sequenceSynthetic 8Gly Val Thr Gly Trp Arg Leu
Cys Glu Arg Ile Leu Ala1 5 109158PRTArtificial sequenceSynthetic
9Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5
10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser
Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile
Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile
Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu
Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe
Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr
Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile
Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu
Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro
Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn145 150
1551011PRTArtificial sequenceSynthetic 10Val Thr Gly Tyr Arg Leu
Phe Glu Glu Ile Leu1 5 101111PRTArtificial sequenceSynthetic 11Val
Ser Gly Trp Arg Leu Phe Lys Lys Ile Ser1 5 1012155PRTArtificial
sequenceSynthetic 12Met Lys His His His His His His Val Phe Thr Leu
Asp Asp Phe Val1 5 10 15Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu
Asp Gln Val Leu Glu 20 25 30Gln Gly Gly Val Ser Ser Leu Leu Gln Asn
Leu Ala Val Ser Val Thr 35 40 45Pro Ile Met Arg Ile Val Arg Ser Gly
Glu Asn Ala Leu Lys Ile Asp 50 55 60Ile His Val Ile Ile Pro Tyr Glu
Gly Leu Ser Ala Asp Gln Met Ala65 70 75 80Gln Ile Glu Glu Val Phe
Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95Phe Lys Val Ile Leu
Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110Pro Asn Lys
Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125Phe
Asp Gly Lys Lys Ile Thr Thr Thr Gly Thr Leu Trp Asn Gly Asn 130 135
140Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp145 150
1551311PRTArtificial sequenceSynthetic 13Gly Ser Met Leu Phe Arg
Val Thr Ile Asn Ser1 5 101422PRTArtificial sequenceSynthetic 14Gly
Ser Met Leu Phe Arg Val Thr Ile Asn Ser Val Ser Gly Trp Arg1 5 10
15Leu Phe Lys Lys Ile Ser 201513PRTArtificial sequenceSynthetic
15Gly Lys Met Leu Phe Arg Val Thr Ile Asn Ser Trp Lys1 5
101613PRTArtificial sequenceSynthetic 16Gly Arg Met Leu Phe Arg Val
Thr Ile Asn Ser Trp Arg1 5 101713PRTArtificial sequenceSynthetic
17Gly Lys Leu Leu Phe Val Val Val Ile Glu Lys Tyr Lys1 5
101813PRTArtificial sequenceSynthetic 18Gly Arg Leu Leu Phe Val Val
Val Ile Glu Arg Tyr Arg1 5 101913PRTArtificial sequenceSynthetic
19Lys Lys Met Leu Phe Arg Val Thr Ile Gln Lys Trp Lys1 5
102013PRTArtificial sequenceSynthetic 20Arg Arg Met Leu Phe Arg Val
Thr Ile Gln Arg Trp Arg1 5 102113PRTArtificial sequenceSynthetic
21Val Ser Val Ser Gly Trp Arg Leu Phe Lys Lys Ile Ser1 5
102213PRTArtificial sequenceSynthetic 22Val Ser Val Ser Gly Trp Arg
Leu Phe Arg Arg Ile Ser1 5 102311PRTArtificial sequenceSynthetic
23Val Ser Gly Trp Arg Leu Phe Arg Arg Ile Ser1 5
102413PRTArtificial sequenceSynthetic 24Asp Lys Leu Leu Phe Thr Val
Thr Ile Glu Lys Tyr Lys1 5 102513PRTArtificial sequenceSynthetic
25Asp Arg Leu Leu Phe Thr Val Thr Ile Glu Arg Tyr Arg1 5
102614PRTArtificial sequenceSynthetic 26Gly Lys Met Leu Phe Arg Val
Thr Ile Asn Ser Trp Lys Cys1 5 102714PRTArtificial
sequenceSynthetic 27Gly Arg Met Leu Phe Arg Val Thr Ile Asn Ser Trp
Arg Cys1 5 102814PRTArtificial sequenceSynthetic 28Gly Lys Leu Leu
Phe Thr Val Thr Ile Glu Lys Tyr Lys Cys1 5 102914PRTArtificial
sequenceSynthetic 29Gly Arg Leu Leu Phe Thr Val Thr Ile Glu Arg Tyr
Arg Cys1 5 103014PRTArtificial sequenceSynthetic 30Lys Lys Met Leu
Phe Arg Val Thr Ile Gln Lys Trp Lys Cys1 5 103114PRTArtificial
sequenceSynthetic 31Arg Arg Met Leu Phe Arg Val Thr Ile Gln Arg Trp
Arg Cys1 5 103214PRTArtificial sequenceSynthetic 32Val Ser Val Ser
Gly Trp Arg Leu Phe Lys Lys Ile Ser Cys1 5 103314PRTArtificial
sequenceSynthetic 33Val Ser Val Ser Gly Trp Arg Leu Phe Arg Arg Ile
Ser Cys1 5 103412PRTArtificial sequenceSynthetic 34Val Ser Gly Trp
Arg Leu Phe Arg Arg Ile Ser Cys1 5 103514PRTArtificial
sequenceSynthetic 35Asp Lys Leu Leu Phe Thr Val Thr Ile Glu Lys Tyr
Lys Cys1 5 103614PRTArtificial sequenceSynthetic 36Asp Arg Leu Leu
Phe Thr Val Thr Ile Glu Arg Tyr Arg Cys1 5 103712PRTArtificial
sequenceSynthetic 37Val Ser Gly Trp Arg Leu Phe Arg Arg Ile Ser
Cys1 5 103814PRTArtificial sequenceSynthetic 38Gly Arg Met Leu Phe
Arg Val Thr Ile Asn Ser Trp Arg Cys1 5 103914PRTArtificial
sequenceSynthetic 39Gly Arg Leu Leu Phe Thr Val Thr Ile Glu Arg Tyr
Arg Cys1 5 10
* * * * *