SITE-SPECIFIC LABELING OF AFFINITY TAGS IN FUSION PROTEINS
First Claim
1. A staining solution for detecting fusion proteins comprising an affinity tag, wherein said staining solution comprises:
- a) a fluorescent compound capable of selectively binding, directly or indirectly, to said affinity tag, wherein said fluorescent compound comprises a fluorophore; and
,a. a buffer;
with the proviso that the fluorescent compound does not comprise an antibody or fragment thereof.
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Accused Products
Abstract
The present invention provides methods and fluorescent compounds that facilitate detecting and labeling of a fusion protein by being capable of selectively binding to an affinity tag. The fluorescent compounds have the general formula A(B)n, wherein A is a fluorophore, B is a binding domain that is a charged chemical moiety, a protein or fragment thereof and n is an integer from 1-6 with the proviso that the protein or fragment thereof not be an antibody or generated from an antibody. The present invention provides specific fluorescent compounds and methods used to detect and label fusion proteins that contain a poly-histidine affinity tag. These compounds have the general formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain, m is an integer from 1 to 4 and n is an integer from 1 to 6. The acetic acid groups interact directly with the positively charged histidine residues of the affinity tag to effectively label and detect a fusion protein containing such an affinity tag when present in an acidic or neutral environment.
101 Citations
52 Claims
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1. A staining solution for detecting fusion proteins comprising an affinity tag, wherein said staining solution comprises:
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a) a fluorescent compound capable of selectively binding, directly or indirectly, to said affinity tag, wherein said fluorescent compound comprises a fluorophore; and
,a. a buffer; with the proviso that the fluorescent compound does not comprise an antibody or fragment thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 14, 15, 16, 17, 18, 19, 20, 36, 37, 38, 39, 40, 41, 42)
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8. A staining solution for detecting fusion proteins comprising a poly-histidine affinity tag, wherein said staining solution comprises:
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a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6; and
,b) a buffer having a pH of about 5 to 6.9 and comprising an acceptable counter ion with the proviso that said binding domain does not comprise an antibody or fragment thereof. - View Dependent Claims (9, 10, 11, 12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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31. The method according to claim 27, wherein said fluorophore is a benzofuran and said compound is selected from the group consisting of
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32. The method according to claim 27, wherein said fluorophore is a borapolyazaindacene and said compound is selected from the group consisting of
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33. The method according to any one of claims 30, 31 or 32, wherein said compound binds directly to said affinity tag of said fusion protein.
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34. The method according to any one of claims 30, 31 or 32, wherein said buffer further comprises a metal ion and said compound indirectly binds said affinity tag by forming a ternary complex.
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35. The method according to claim 34 wherein said metal ion is nickel or cobalt.
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- 43. A fluorescent compound having formula A(L)m(B)n, wherein A is a fluorophore selected from the group consisting of borapolyazaindacene and coumarin, L is a linker, B is an acetic acid binding domain wherein said fluorescent compound contains at least three acetic acid groups that are capable of binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6.
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48. A composition comprising;
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a) a fluorescent compound capable of selectively binding, directly or indirectly, to affinity tag containing fusion protein, wherein said fluorescent compound comprises a fluorophore; and
,b) a fusion protein comprising an affinity tag, provided said fluorescent compound does not comprise an antibody or fragment thereof. - View Dependent Claims (49, 50, 51, 52)
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Specification