U.S. patent application number 10/661451 was filed with the patent office on 2005-10-20 for site-specific labeling of affinity tags in fusion proteins.
Invention is credited to Diwu, Zhenjun, Gee, Kyle, Hart, Courtenay, Haugland, Richard, Leung, Wai-Yee, Patton, Wayne, Rukavishnikov, Aleksey.
Application Number | 20050233307 10/661451 |
Document ID | / |
Family ID | 31997971 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233307 |
Kind Code |
A1 |
Gee, Kyle ; et al. |
October 20, 2005 |
Site-specific labeling of affinity tags in fusion proteins
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.
Inventors: |
Gee, Kyle; (Springfield,
OR) ; Hart, Courtenay; (Eugene, OR) ; Leung,
Wai-Yee; (Eugene, OR) ; Patton, Wayne;
(Eugene, OR) ; Rukavishnikov, Aleksey; (Eugene,
OR) ; Haugland, Richard; (Eugene, OR) ; Diwu,
Zhenjun; (Sunnyvale, CA) |
Correspondence
Address: |
KOREN ANDERSON
MOLECULAR PROBES, INC.
29851 WILLOW CREEK ROAD
EUGENE
OR
97402-9132
US
|
Family ID: |
31997971 |
Appl. No.: |
10/661451 |
Filed: |
September 12, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60410612 |
Sep 12, 2002 |
|
|
|
60458472 |
Mar 28, 2003 |
|
|
|
Current U.S.
Class: |
435/4 ;
436/86 |
Current CPC
Class: |
A61K 49/0021 20130101;
A61K 49/0039 20130101; C07D 311/16 20130101; G01N 33/533 20130101;
C07F 5/022 20130101; A61K 49/0041 20130101; G01N 33/52 20130101;
G01N 33/582 20130101; C07D 311/12 20130101; A61K 49/0052 20130101;
C07D 311/18 20130101 |
Class at
Publication: |
435/004 ;
436/086 |
International
Class: |
C12Q 001/00; G01N
033/00 |
Claims
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, b) a buffer; with the proviso that
the fluorescent compound does not comprise an antibody or fragment
thereof.
2. The staining solution according to claim 1, wherein said
fluorescent compound is capable of selectively binding to a
poly-histidine, GST, poly-arginine or Glu-Glu affinity tags.
3. The staining solution according to claim 1, wherein said
fluorescent compound is according to formula A(L)m(B)n wherein A is
a fluorophore, L is a linker, B is binding domain, m is an integer
from 1 to 4 and n is an integer from 1 to 6.
4. The staining solution according to claim 3, wherein said
fluorophore is selected from the group consisting of xanthene,
coumarin, cyanine, acridine, anthracene, benzofuran, indole and
borapolyazaindacene.
5. The staining solution according to claim 4, wherein said
fluorescent compound comprises glutathione as a binding domain and
xanthene as a fluorophore.
6. The staining solution according to claim 4, wherein said binding
domain is an acetic acid binding domain.
7. The staining solution according to claim 6, wherein said acetic
acid binding domain is capable of selectively binding, directly or
indirectly, to a poly-histidine or a poly-arginine affinity
tag.
8. A staining solution for detecting fusion proteins comprising a
poly-histidine affinity tag, wherein said staining solution
comprises: 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.
9. The staining solution according to claim 8, wherein said buffer
comprises a salt.
10. The staining solution according to claim 9, wherein said
fluorophore is selected from the group consisting of xanthene,
coumarin, cyanine, acridine, anthracene, benzofuran, indole and
borapolyazaindacene.
11. The staining solution according to claim 10, wherein said
buffer has a pH of about 6.5.
12. The staining solution according to claim 11, wherein said
buffer further comprises a metal ion selected from the group
consisting of nickel and cobalt.
13. The staining solution according to claim 12, wherein said
staining solution comprises nickel ions at a final concentration of
about 1 .mu.M to 150 .mu.M.
14. A method for selectively detecting an affinity tag containing
fusion protein in a sample, said method comprising the steps of a)
contacting said sample with a staining solution comprising a buffer
and a fluorescent compound capable of selectively binding, directly
or indirectly, to said affinity tag, wherein said fluorescent
compound comprises a fluorophore; and, b) illuminating said
fluorescent compound whereby said fusion protein is detected with
the proviso that said fluorescent compound does not comprise an
antibody or fragment thereof.
15. The method according to claim 14, wherein said method further
comprises first immobilizing said sample on a solid or semi-solid
matrix.
16. The method according to claim 14, wherein said affinity tag is
selected from the group consisting of poly-histidine, GST,
poly-arginine and Glu-Glu affinity tags.
17. The method according to claim 16, wherein said fluorophore is
selected from the group consisting of a xanthene, coumarin,
cyanine, acridine, anthracene, benzofuran, indole and
borapolyazaindacene.
18. The method according to claim 17, wherein said compound
comprises formula A(B)n wherein A is a fluorophore, B is a binding
domain that is a chemical moiety, protein or fragment thereof
capable of selectively binding said affinity tag and n is an
integer from 1 to 6.
19. The method according to claim 18, wherein said chemical moiety
is an acetic acid binding domain.
20. The method according to claim 19, wherein said buffer further
comprises an indirect binding reagent capable of forming a complex
between said affinity peptide and said binding moiety.
21. A method for detecting a poly-histidine affinity tag containing
fusion protein in a sample, said method comprising the steps of: i)
immobilizing said sample on a solid or semi-solid matrix; ii)
optionally contacting said sample of step i) with a fixing
solution; iii) contacting said sample of step i) or ii) with a
staining solution comprising a buffer and a fluorescent compound
capable of selectively binding directly or indirectly to said
affinity tag, wherein said fluorescent compound comprises a
fluorophore; iv) incubating said staining solution and said sample
for sufficient time to allow said compound to associate either
directly or indirectly with said poly-histidine affinity tag; v)
illuminating fluorophore of said staining solution with a suitable
light source whereby said fusion protein is detected.
22. The method according to claim 21, wherein said buffer has a pH
of about 6.5.
23. The method according to claim 22, wherein said buffer comprises
a salt.
24. The method according to claim 23, wherein said buffer has a pKa
of about 6.0 to about 7.5.
25. The method according to claim 24, wherein said fluorophore is
selected from the group consisting of xanthene, cyanine, coumarin,
acridine, anthracene, benzofuran, borapolyazaindacene and
derivative thereof.
26. The method according to claim 25, wherein fluorescent compound
of said staining solution comprises at least three acetic acid
groups.
27. The method according to claim 26, wherein immobilizing said
sample comprises electrophoretically separating on a polymeric
gel.
28. The method according to claim 27, wherein said fixing solution
comprises an alcohol.
29. The method according to claim 28, wherein said method further
comprises contacting said gel with a total protein stain.
30. The method according to claim 27, wherein said fluorophore is a
coumarin and said compound is selected from the group consisting of
28and salts thereof.
31. The method according to claim 27, wherein said fluorophore is a
benzofuran and said compound is selected from the group consisting
of 2930and salts thereof.
32. The method according to claim 27, wherein said fluorophore is a
borapolyazaindacene and said compound is selected from the group
consisting of 3132
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.
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.
35. The method according to claim 34 wherein said metal ion is
nickel or cobalt.
36. A kit for detecting an affinity tag containing fusion protein,
wherein said kit comprises; a staining solution comprising a buffer
and a fluorescent compound capable of selectively binding, directly
or indirectly, to an affinity tag, wherein the fluorescent compound
comprises a fluorophore; with the proviso that the fluorescent
compound does not comprise an antibody or fragment thereof.
37. The kit according to claim 36, wherein said kit further
comprises, alone or in combination, molecular weight markers,
fixing solution, wash solution and an additional detection
reagent.
38. The kit according to claim 36, wherein said additional
detection reagent is a total protein stain.
39. The kit according to claim 36, wherein said fluorescent
compound comprises a binding domain and a fluorophore selected from
the group consisting of a xanthene, cyanine, coumarin, acridine,
anthracene, benzofuran, borapolyazaindacene and derivative
thereof.
40. The kit according to claim 39, wherein said fluorescent
compound is according to 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 about 1 to 4 and n is an integer from about 1 to
6 wherein said fluorescent compound comprises at least three acetic
acid groups.
41. The kit according to claim 40 wherein said buffer has a pH
between about 5 to about 6.9 and said buffer optionally comprises a
metal ion selected from the group consisting of nickel and
cobalt.
42. The kit according to claim 39, wherein said binding domain is
glutathione.
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.
44. The compound according to claim 43, wherein said linker is
selected from the group consisting of
--(CH.sub.2).sub.eC(X)NH(CH.sub.2)C(NHC(X)(C-
H.sub.2).sub.e).sub.d--,
--((C.sub.6R'.sub.4)O).sub.d(CH.sub.2).sub.e(C(X)-
NH(CH.sub.2).sub.e)(NH).sub.dC(X)NH(C.sub.6R".sub.4)(CH.sub.2).sub.e--
and --(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4) wherein X is O or
S, d is 0 or 1, e is 1 to 6, f is 2 or 3, and R" is independently
H, halogen, alkoxy or alkyl.
45. The compound according to claim 44, wherein said acetic acid
binding domain is selected from the group consisting of
.sup.-O.sub.2CCH(R)N(CH.s- ub.2CO.sup.-.sub.2).sub.2,
--N(CH.sub.2CO.sub.2).sub.2 and
(CH.sub.2CO.sup.-.sub.2).sub.ZN[(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2)].s-
ub.T(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2).sub.Z wherein Z is 1 or
2, S is 1 to 5, T is O to 4 and R is said linker.
46. The compound according to claim 45, wherein said fluorophore is
a borapolyazaindacene and said compound is selected from the group
consisting of 3334and salts thereof wherein R.sup.30 may be the
same or different and is selected from the group consisting of
hydrogen, salt ion, --CH.sub.2OCOR.sup.41 and an electron pair
wherein R.sup.41 is an alkyl group.
47. The compound according to claim 45, wherein said fluorophore is
a coumarin and said compound is selected from the group consisting
of 35and salts thereof wherein R.sup.10 may be the same or
different and is selected from the group consisting of hydrogen,
salt ion, --CH.sub.2OCOR.sup.41 and an electron pair wherein
R.sup.41 is an alkyl group.
48. A composition comprising; 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.
49. The composition according to claim 48, wherein said fluorescent
compound comprises a binding domain and a fluorophore selected from
the group consisting of xanthene, cyanine, coumarir, acridine,
anthracene, benzofuran, borapolyazaindacene and derivative
thereof.
50. The composition according to claim 49 wherein said fluorescent
compound is according to formula A(L)m(B)n wherein A is a
fluorophore, B is an acetic acid binding domain wherein said
compound comprises at least three acetic acid groups that are
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.
51. The composition according to claim 50, wherein said composition
further comprises a metal ion selected from the group consisting of
nickel and cobalt.
52. The composition according to claim 49, wherein said binding
domain is glutathione.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel compositions and
methods for the detection and isolation of fusion proteins
comprising affinity tag sequences. The invention has applications
in the fields of molecular biology and proteomics.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to fluorescent compounds that
have selective affinity, and bind with specificity to affinity
tag-containing fusion proteins. Such compounds being particularly
useful for the detection, site-specifically labeling and monitoring
of desired recombinant fusion proteins.
[0003] Typically, recombinant fusion proteins comprise a synthetic
leader peptide or protein fragment linked to independently derived
polypeptides. In 1965 it was demonstrated that an amino acid
sequence not normally part of a given operon can be inserted within
the operon and be controlled by the operon (Jacob, F. et al. (1965)
J. Mol. Biol. 13, 704). Therefore, the leader sequence of
recombinant fusion proteins can facilitate protein expression,
detection and purification by providing, for example, enzymatic
activity enabling identification of fusion proteins, an amino acid
sequence recognized by cellular secretory mechanism, or a sequence
having distinctive chemical or antigenic characteristics useful in
purifying and detection of the fusion protein by ion exchange,
reverse phase, immunoaffinity and affinity chromatographic media.
In general, polyanionic peptides and polycationic peptides bind to
ion-exchangers, hydrophobic peptides bind to reverse-phase media
and peptides that are immunogenic can be bound by antibodies.
[0004] Immobilized metal-ion affinity chromatography (IMAC) relies
upon the interaction of exposed histidine and cysteine residues on
proteins with certain transition metals, such as Ni.sup.2+,
Co.sup.2+, Zn.sup.2+, Cu.sup.2+ and Fe.sup.3+ (Porath, J., et al.
(1975) Nature 258:598-599; Winzerling, J., et al. (1992) Methods
4:4-13; Yip, T. and Hutchens, T. (1994) Molecular Biotechnol.
1:151-164). Protein interaction with immobilized metal ions is a
selective and versatile, high-affinity adsorption procedure. The
basic principles of IMAC are commonly exploited to facilitate the
purification of recombinant proteins.
[0005] The poly-histidine affinity tag is a transition
metal-binding peptide sequence comprising a string of four to ten
histidine residues. When a DNA sequence corresponding to the
poly-histidine affinity tag is fused in frame with a gene, the
resulting fusion protein can readily be purified by IMAC using a
nickel- or cobalt-charged resin. Though a variety of fusion
affinity tags have been developed over the years, the
poly-histidine affinity tag is popular because it requires minimal
addition of extra amino acids to the recombinant protein, rarely
interferes with protein folding, is poorly immunogenic and allows
for rapid purification of the target protein by IMAC.
[0006] Unfortunately, the detection of poly-histidine affinity tag
containing fusion proteins after electrophoresis usually requires
multiple time-consuming steps, including transfer of the gel to a
membrane, blocking of unoccupied sites on the membrane with protein
or detergent solutions, incubation with a poly-histidine affinity
tag-binding agent (primary antibody, biotin-nitrilotriacetic acid
or HRP-nitrilotriacetic acid), incubation with a secondary
detection agent (antibody-reporter enzyme conjugate,
streptavidin-reporter enzyme conjugate), and incubation with a
visualization reagent (calorimetric, fluorogenic or
chemiluminescent reagent). Specifically, biotinylated
nitrilotriacetic acid (NTA) has been used in combination with
streptavidin-horseradish peroxidase or streptavidin-alkaline
phosphatase conjugates and chemiluminescent or calorimetric
substrates in order to detect poly-histidine affinity tag
containing fusion proteins after electroblotting (Hochuli, E. and
Piesecki, S. (1992) Methods 4: 68-72; O'Shannessy, D., etal. (1995)
Anal. Biochem. 229:119-124; McMahan, S. and Burgess, R. (1996) Anal
Biochem. 236: 101-106). In addition, direct reporter
enzyme-nitrilotriacetate-nickel conjugates have been employed for
detection of poly-histidine affinity tag containing fusion proteins
on electroblots (Botting, C. and Randall, R. (1995) BioTechniques
19: 362-363; Jin, L., et al. (1995) Anal. Biochem. 229: 54-60).
Similarly, colloidal gold with nitrilotriacetic acid conjugated to
its surface has been employed to detect poly-histidine affinity tag
containing fusion proteins on blots after a silver enhancement step
(Hainfeld, J., et al. (1999) J. Struct. Biol. 127: 185-198).
Finally, though poly-histidine affinity tag is not particularly
immunogenic, a number of high affinity monoclonal antibodies
specific to the peptide have been generated to detect affinity tag
containing fusion proteins by standard electroblotting methods
(Zentgraf, H., et al. (1995) Nucleic Acids Res. 23: 3347-3348;
Pogge von Strandmann, E., et al. (1995) Protein Eng. 8: 733-735;
Lindner, P., et al. (1997) BioTechniques 22: 140-149).
[0007] Examples of immunogenic affinity tags include protein A,
c-myc (Roth et al, (1991) J. Cell Biol. 115:587-596), myc
(EQKLISEEDL; Evan G I, et al. (1985) Mol. Cell Biol. 5:3610-3616;
Munro S. and Pelham H R B, (1987) Cell 48:899-907; Bodjigin J. and
Nathans J., (1994) 269:14715-14727; Smith D J, (1997) BioTechniques
23:116-120) FLAG.RTM. (Hopp T. P. et al. (1988) Biotechnology
6:1204; Prickett, K. S. et al. (1989) BioTechniques 7:580-589;
Gerard N P and Gerard C, (1990) Biochemistry 29:9274-9281; Einhauer
A. and Jungbauer A. (2001) J. Biochem Biophys. Methods 49:455-465;
U.S. Pat. Nos. 4,703,004; 4,851,341 and 5,011,912), GST
(Glutathione-S-transferase), HA, derived from the influenza
hemagglutinin protein (Wilson I A, et al., (1984) Cell, 37:767;
Field J. et al. Mol. Cell Biol. (1988) 8:2159-2165; Xu Y, et al.
(2000) Mol Cell Biol. 20:2138-2146), IRS (RYIRS; Liang T C et al.
(1996) 329:208-214; Luo W et al (1996) Arch. Biochem. Biophys.
329:215-220), AU1 and AU5 (DTYRYI and TDFLYK; Lim P S et al. (1990)
J. Infect. Dis. 162:1263-1269; Goldstein D J et al. (1992)
190:889-893; Koralnik I J et al. (1993) J. Virol. 67:2360-2366),
glu-glu (a 9 amino acid epitope from polyoma virus medium T
antigen, EEEEYMPME; Grussenmeyer, T. et al. (1985) PNAS. USA
82:7952-7954; Rubinfeld. B. et al. (1991) Cell 65:1033-1042), KT3
(an 11 amino acid epitope from the SV40 large T antigen,
KPPTPPPEPET; MacArthur H. and Walter G. (1984) J. Virol.
52:483-491; Martin G A et al. (1990) 63:843-849; Di Paolo G et al.
(1997) 272:5175-5182), T7 (an 11 amino acid leader peptide from T7
major capsid protein), S-TAG, HSV (an 11 amino acid peptide from
herpes simplex virus glycoprotein D), VSV-G (an 11 amino acid
epitope from the carboxy terminus of vesicular stomatitis virus
glycoprotein, YTDIEMNRLGK; Kreis T. (1986) EMBO J. 5:931-941;
Turner J R et al (1996) 271:7738-7744), Anti-Xpress (8 amino acid
epitope, DLYDDDK), and VS (14 amino acid epitope from paramoxyvirus
SV5, GKPIPNPLLGLDST).
[0008] Typically, immunogenic affinity tags are detected with
labeled antibodies wherein the label can be an enzyme, fluorophore,
hapten or any label known to one skilled in the art and the
antibodies, directly or indirectly, detect the affinity containing
fusion protein. Immunogenic affinity tags can also be detected in a
multistep assay using ruthenium labeled anti-affinity tag
antibodies that produce electrochemiluminescenc- e (ECL)
(ORIGEN.RTM., U.S. Pat. Nos. 5,310,687; 5,714,089; 5,453,356;
6,140,138; 5,804,400 and 5,238,808) indicating the presence of the
affinity tag. Electrochemiluminescence is the process by which
light generation occurs when a low voltage is applied to an
electrode, triggering a cyclical oxidation and reduction reaction
of a ruthenium metal ion bound to the compound to be detected. The
ruthenium labeled antibody is captured on a solid surface by the
affinity tag, a second oxidation reaction component, tripropylamine
(TPA), is introduced into the cell and a voltage is applied. The
TPA reduces the ruthenium, which receives the electron in an
excited state and then decays to the ground state releasing a
photon in the process.
[0009] The FLAG.RTM. affinity tag was designed in conjunction with
antibodies for the purpose of detection and purification of fusion
proteins (Hopp T. P. et al. (1988) Biotechnology 6:1204; Prickett,
K. S. et al. (1989) BioTechniques 7:580-589, supra). As such, the
use of anti-FLAG.RTM. antibodies are widely used to detect and
purify FLAG.RTM. affinity tag containing fusion proteins. The
FLAG.RTM. sequence typically consists of DYKDDDDK, D=Asp, Y=Tyr and
K=Lys, but any combination of 3 to 6 aspartic or glutamic acid
residues is also considered a FLAG.RTM. sequence. The sequence is
hydrophilic and highly immunogenic. The FLAG.RTM. affinity tag has
effectively been used in various expression systems for the
detection and purification of recombinant fusion proteins (Brizzard
et al. (1994) BioTechniques 16:730-735; Lee et al. (1994) Nature
372:739-746; Xu et al. (1993) Development 117:1223-1237; Dent et
al. (1995) Mol. Cell Biol. 15:4125-4135; Ritchie et al. (1999)
BioChem Journal 338:305-10.) Recently, the FLAG.RTM. affinity tag
was used to detect fusion proteins wherein the use of antibodies
was not employed (Buranda T. et al. (2001) Anal. Biochemistry
298:151-162). The FLAG.RTM. sequence was synthesized with
fluorescein and/or biotin as a label and tag, respectively, wherein
the peptides were bound to streptavidin beads and the fluorescein
was detected using flow cytometry.
[0010] While antibodies against GST are available for both
purification and detection (Molecular Probes, Inc., Eugene, Oreg.)
the affinity tag is typically purified using glutathione resin
(U.S. Pat. Nos. 5,654,176; 6,303,128 and 6,013,462). Glutathione is
a ubiquitous tripeptide that binds with high affinity to the GST
enzyme.
[0011] An affinity tag that is not generally immunogenic and does
not readily bind metal ions or chemical moieties includes
calmodulin-binding peptides (U.S. Pat. Nos. 5,585,475; 6,316,409
and 6,117,976). These affinity tags are routinely purified using
columns wherein beads are covalently attached to calmodulin. In the
presence of calcium the calmodulin protein binds the calmodulin
affinity tag with high affinity because calcium induces a
conformational change in calmodulin increasing the affinity of the
protein for the affinity tag. Calmodulin affinity tags are
advantageous in certain applications because the captured fusion
protein can be eluted from a column using a metal chelating moiety
instead of harsh denaturing conditions.
[0012] Another affinity tag that is not generally immunogenic
includes the binding site for the FlAsH reagent, CCXXCC wherein X
is an amino acid other than cysteine (Griffin et al (2000) Methods
in Enzymology 327:565-578; Griffin et al (1998) Science
281:269-272; Thorn et al (2000) Protein Science 9:213-217). The
FlAsH reagent is a fluorescein molecule that has been substituted
by two arsenical groups such that the reagent interacts with the
.alpha.-helical structure of the CCXXCC sequence (Adams et al
(2002) Journal of American Chemical Society 124: 6063-6076). For
binding to occur the thiols of the cysteine residues must not be
disulfide bonded or chelated by a metal ion. Thus, the FlAsH
reagent is typically used to label proteins in vivo due to these
limitations for in vitro labeling. Therefore a reducing agent must
be used for binding to occur and a buffer must be free of metal
ions.
[0013] The fluorescent compounds and methods of the present
invention have been developed for the fluorescence detection of
affinity tag containing fusion proteins directly in polymeric gels
(with or without sodium dodecyl sulfate (SDS)), without the
requirement for electroblotting, blocking, reporter enzymes or
secondary detection reagents. These present fluorescent compounds
are advantages over FlAsH wherein a reducing agent is not required
and a metal ion may be present in the buffer solution. These
compounds take advantage of the charged residues of the affinity
tag wherein the binding domains of the present invention are
covalently attached to a fluorophore for selective detection of a
wide range of affinity tag containing fusion proteins. These
compounds and methods of the present invention provide a
significant improvement over the prior art for detecting,
monitoring and quantitating affinity tag containing fusion
proteins.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods and fluorescent
compounds that specifically and selectively bind to affinity tags
of fusion proteins. The compounds of the present invention
facilitate detecting and labeling of a fusion protein by being
capable of selectively binding to an affinity tag. The methods for
detecting a fusion protein containing an affinity tag comprises
contacting a sample with a staining solution and then illuminating
the sample whereby the fusion protein is detected. The staining
solution comprises a fluorescent compound and a buffer wherein the
buffer optionally comprises a metal ion. The fluorescent compounds,
as used herein, are defined as a compound that is capable of
selectively binding, directly or indirectly to an affinity tag.
[0015] 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 binding domain of
the fluorescent compound may bind directly or indirectly to the
affinity tag. When the fluorescent compound binds directly, the
charged chemical moiety or protein of the binding domain interacts
directly to form a non-covalent bond between the fluorescent
compound and the affinity tag of the fusion protein. When the
compounds of the present invention bind indirectly, a metal ion
facilitates the indirect binding by having affinity for both the
charged amino acid residues of the affinity tag and the binding
domain of the fluorescent compound. The indirect binding of the
fluorescent compound results in a ternary complex of the
fluorescent compound, metal ion and affinity tag of the fusion
protein.
[0016] The present invention provides specific fluorescent
compounds and methods used to detect and label fusion proteins that
contain a poly-histidine affinity tag or a poly-arginine affinity
tag. These compounds have the general formula A(L)m(B)n wherein A
is a fluorophore, L is a linker, B is a binding domain, m is an
integer from 1 to 4 and n is an integer from 1 to 6. The linker
functions to covalently attach the fluorophore to the binding
domain wherein the resulting fluorescent compound contains an
acetic acid binding domain. The acetic acid groups interact
directly with the positively charged histidine or arginine residues
of the affinity tag to effectively label and detect a fusion
protein containing such an affinity tag when present in a slightly
acidic or neutral environment. Alternatively, the acetic acid
groups of the fluorescent compound have an affinity for the metal
ions nickel and cobalt wherein the metal ions also have affinity
for the poly-histidine affinity tag of the fusion peptide. This
indirect labeling and detection of the fusion protein may in
certain circumstances be as effective as the direct method that
does not utilize the metal ions for labeling and detecting fusion
proteins containing poly-histidine affinity tags. The fluorescent
compounds of the present invention that have an affinity for
poly-histidine affinity tags effectively bind non-covalently when
present in a moderately acidic or neutral environment, preferably
in a buffer with a pH about 5 to 7.0.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Shows the detection of a poly-histidine affinity tag
containing fusion protein (urate oxidase) labeled with Compound 2
in a staining solution containing (1A) nickel ions and (2B) without
nickel ions. In this particular assay, Compound 2 demonstrates an
increased sensitivity for the poly-histidine affinity tag in the
absence of nickel ions.
[0018] FIG. 2: Shows the detection of a (2A) poly-histidine
affinity tag containing fusion protein (Oligomycin sensitivity
conferring protein; OSCP) labeled with Compound 2 in a staining
solution containing nickel ions followed by the detection of (2B)
total protein using the total protein stain SYPRO.RTM. Ruby. This
assay demonstrates that Compound 2 is selective for the
poly-histidine affinity tag.
[0019] FIG. 3: Shows the detection of a (3A) poly-histidine
affinity tag containing fusion protein (Oligomycin sensitivity
conferring protein; OSCP) labeled with Compound 15 in a staining
solution containing nickel ions followed by the detection of (3B)
total protein using the total protein stain SYPRO.RTM. Ruby. This
assay demonstrates that Compound 15 is selective for the
poly-histidine affinity tag.
[0020] FIG. 4: Shows the detection of GST affinity tag using Texas
Red X-glutathione fluorescent compound on a polyacrylamide gel.
Purified glutathione S-transferase (1 .mu.g) at 24 and 25 mm from
the gel origin (2 peaks) stained with 5 .mu.M Texas Red
X-Glutathione in 50 mM PIPES pH 6.5. Imaged on the Fuji FLA3000 at
532 nm excitation, 580LP filter. See, Example 20.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0021] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a fusion protein" includes a plurality of proteins
and reference to "a fluorescent compound" includes a plurality of
compounds and the like.
[0022] Unless defined otherwise, 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 is related. The
following terms are defined for purposes of the invention as
described herein.
[0023] The term "acetic acid binding domain" as used herein refers
to a domain that contains at least two terminal acetic acid groups,
as defined below. The acetic acid binding domains contain nitrogen
as the point of attachment for the acetic acid groups and the
binding domain is attached to a linker at either a nitrogen or
carbon atom depending on one of the three (I, II or III) formulas
for the binding domain. Specifically, the acetic acid binding
domains have formula (I) O.sub.2CCH(R)N(CH.sub.2CO.su-
p.-.sub.2).sub.2, wherein R is a linker that is covalently bonded
to the methine carbon atom (See, for example Compound 1), or
formula (II) --N(CH.sub.2CO.sub.2.sup.-).sub.2 wherein the linker
is covalently bonded to the nitrogen atom (See, for example
Compound 12).
[0024] Alternatively, the acetic acid binding domain has formula
(III)
(CH.sub.2CO.sup.-.sub.2).sub.ZN[(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2)].s-
ub.T(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2).sub.Z wherein the linker
is attached to a methine carbon or nitrogen atom and Z is 1 or 2, S
is 1 to 5 and T is 0 to 4. In all cases, the acetic acid binding
domain contains at least two acetic acid groups and the nitrogen
atom is the point of attachment for the acetic acid groups.
[0025] The term "acetic acid group" as used herein refers to the
chemical formula (IV) --CH(R)CO.sup.-.sub.2, which includes the
protenated form --CH(R)CO.sub.2H. R is independently H or a Linker,
as defined below. When R is hydrogen the acetic acid group has the
formula --CH.sub.2CO.sup.-.sub.2. When the linker of the
fluorescent compound is attached to a methine carbon of an acetic
acid group then R is the linker. When an acetic acid group is
referred to, it is understood to be a terminal end of a compound,
which allows for the negatively charged carboxy group of the acetic
acid group to freely interact with a positively charged
affinity-binding domain. When acetic acid groups are part of the
binding domain, nitrogen is the point of attachment for the acetic
acid groups. These binding domains are particularly useful for
labeling and detecting poly-histidine affinity tags, e.g.
.sup.-O.sub.2CCH(R)N(CH.sub.2CO.sub.2).sub.2 wherein R is the point
of attachment of the Linker.
[0026] The term "affinity" as used herein refers to the strength of
the binding interaction of two molecules, such as a metal chelating
compound and a metal ion or a positively charged moiety and a
negatively charged moiety.
[0027] The term "affinity tag" as used herein refers to any known
amino acid sequence fused to a protein of interest at either the
amino terminal or carboxy terminal end of the protein (K. Terpe,
Appl. Microbiol. Biotechnol (2003) 60:523-533). Typically, the
affinity tag is used for isolation and or detection purposes. The
"affinity tag" may optionally be in the middle of the protein of
interest such that when the corresponding nucleic acid sequence is
translated the affinity tag is fused in frame into the protein of
interest. The amino acid residues form a peptide that has affinity
for a chemical moiety, a metal ion or a protein. The affinity tag
may have an overall positive, negative or neutral charge; typically
the affinity tag has an overall positive or negative charge.
[0028] The term "affinity-tag-containing-fusion protein" as used
herein refers to a fusion protein that contains a protein of
interest and an affinity tag.
[0029] The term `alkyl` as used herein refers to a straight,
branched or cyclic hydrocarbon chain fragment containing between
about one and about twenty five carbon atoms (e.g. methyl, ethyl
and the like). Straight, branched or cyclic hydrocarbon chains
having eight or fewer carbon atoms will also be referred to herein
as "lower alkyl". In addition, the term "alkyl" as used herein
further includes one or more substitutions at one or more carbon
atoms of the hydrocarbon chain fragment. Such substitutions
include, but are not limited to: aryl; heteroaryl; halogen; alkoxy;
amine (--NH); carboxy and thio.
[0030] The term "aqueous solution" as used herein refers to a
solution that is predominantly water and retains the solution
characteristics of water. Where the aqueous solution contains
solvents in addition to water, water is typically the predominant
solvent.
[0031] The term "B binding domain", "B" and "binding domain" as
used herein refer to a component of the fluorescent compound that
interacts directly or indirectly with the affinity tag of the
fusion protein. The binding domain can be a chemical moiety that
has an overall charge or a protein, provided the protein is not an
antibody or a fragment thereof. The binding domain may be
substituted to adjust the binding affinity, solubility or other
physical properties of the fluorescent compound that the binding
domain is covalently attached to. An important aspect of the
invention is that the binding domain does not contain an arsenic
atom.
[0032] The term "benzofuran" as used herein refers to a fluorophore
generally having the structure below and derivatives thereof. 1
[0033] The benzofuran is typically attached to a
1,2-bis(2-aminophenoxy)et- hane-N,N,N',N'-tetraacetic acid (BAPTA)
metal chelating group by a single covalent bond at any carbon atom
of the BAPTA compound or as a fused ring wherein ring A on the
benzofuran would also be one of the two aromatic carbon rings of
the BAPTA compound. The benzofuran may optionally be further
substituted or unsubstituted as depicted below. 2
[0034] The fluorophore may also be further substituted by
substituents that adjust the binding solubility, spectral
properties or other physical properties of the fluorophore.
[0035] The term "borapolyazaindacene" as used herein refers to a
fluorophore generally having the formula and derivatives thereof
3
[0036] The fluorophore is covalently attached by a linker to at
least one binding domain such as an acetic acid binding domain to
form a compound of the present invention. The fluorophore may also
be further substituted by substituents that adjust the solubility,
spectral properties or other physical properties of the
fluorophore
[0037] The term "buffer" as used herein refers to a system that
acts to minimize the change in acidity or basicity of the solution
against addition or depletion of chemical substances.
[0038] The term "calmodulin" as used herein refers to a binding
domain that when complexed with calcium binds the calmodulin
affinity tag.
[0039] The term "calmodulin affinity tag" as used herein refers to
the amino acid sequence that codes for calmodulin binding peptide
and includes any corresponding peptides disclosed in U.S. Pat. Nos.
5,585,475; 6,117,976 and 6,316,409. The "calmodulin affinity tag"
is fused to a protein of interest for the purposes of detection and
purification.
[0040] The term "coumarin" as used herein refers to a fluorophore
generally having the structure below and derivatives thereof,
wherein A is OR' or N(R').sub.2 wherein R' is hydrogen or alkyl.
4
[0041] The fluorophore is covalently attached by a linker to at
least one binding domain, such as an acetic acid binding domain, to
form a compound of the present invention. The fluorophore may also
be further substituted by substituents that adjust the solubility,
spectral properties or other physical properties of the
fluorophore.
[0042] The term "complex" as used herein refers to the association
of two or more molecules, usually by non-covalent bonding, e.g.,
the association between the negatively charged acetic acid groups
and the positively charged histidine residues of a poly-histidine
affinity tag.
[0043] The term "detectable response" as used herein refers to an
occurrence of, or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an occurrence of a signal
wherein the fluorophore is inherently fluorescent and does not
produce a significant change in signal upon binding to a metal ion
or biological compound. Alternatively, the detectable response is
an optical response resulting in a change in the wavelength
distribution patterns or intensity of absorbance, fluorescence or a
change in light scatter, fluorescence lifetime, fluorescence
polarization, or a combination of the above parameters.
[0044] The term "direct binding" as used herein refers to binding
of the fluorescent compound to the affinity tag of the fusion
protein with the proviso that a metal ion does not comprise the
resulting complex. Typically the charged binding domain of the
fluorescent compound has an affinity for the charged amino acid
residues of the affinity tag wherein a stable non-covalent bond is
formed between the compound and peptide.
[0045] The term "FLAG affinity tag" as used herein refers to the
amino acid sequence DYKDDDDK and any corresponding peptide
disclosed in U.S. Pat. Nos. 4,851,341 and 5,011,912, wherein the
FLAG affinity tag is fused to a protein of interest.
[0046] The term "fluorescent compound" as used herein refers to a
compound with the general formula A(B)n wherein A is a fluorophore,
B is a binding domain comprising a chemical moiety, protein or
fragment thereof that is capable of binding, directly or
indirectly, to the affinity tag of the fusion protein wherein n is
an integer from about 1 to about 6, with the proviso that the
fluorescent compound does not comprise an antibody or fragment
thereof. When the binding domain is a chemical moiety the
fluorescent compound has the general formula A(L)m(B)n wherein L is
a Linker that covalently attaches the fluorophore to the binding
domain. The fluorescent compound of the present invention
effectively non-covalently attaches a fluorophore to the fusion
protein at the site of the affinity tag.
[0047] The term "fluorophore" as used herein refers to a compound
that is inherently fluorescent or demonstrates a change in
fluorescence upon binding to a biological compound or metal ion,
i.e., fluorogenic. Numerous fluorophores are known to those skilled
in the art and include, but are not limited to, coumarin, cyanine,
acridine, anthracene, benzofuran, indole, borapolyazaindacene and
xanthenes including fluorescein, rhodamine and rhodol as well as
other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR
PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS
(9.sup.th edition, CD-ROM, 2002).
[0048] The term "fusion protein" as used herein refers to a protein
hybrid containing an affinity tag and a protein of interest or any
amino acid sequence of interest. The affinity tag may be directly
linked or indirectly linked to the fusion protein. When the
affinity tag is indirectly linked there is preferably a cleavage
site between the affinity tag and the protein of interest that
facilitates recovery of the protein of interest free from the
affinity tag. When a fusion protein containing a cleavage site
comes into contact with an appropriate protease that is specific
for the cleavage site, such as enterokinase, the fusion protein is
cleaved into two polypeptides: the affinity tag and the protein of
interest.
[0049] The term "Glu-Glu affinity tag" as used herein refers to the
amino acid sequence EEEEYMPME or a fragment thereof that is fused
to a protein of interest, either at an end or within the
protein.
[0050] The term "glutathione" as used herein refers to a
tripeptide, or derivative thereof, that specifically binds to the
GST affinity tag and when part of a fluorescent compound of the
present invention represents the binding domain of the fluorescent
compound. Typically, "glutathione" refers to the tripeptide
yglutamylcysteinylglycine, Glu-(Cys-Gly).
[0051] The term "GST affinity tag" as used herein refers to an
amino acid sequence that encodes for all or part of glutathione
S-transferase including any corresponding polypeptides disclosed in
U.S. Pat. No. 5,654,176, that is fused to a protein of
interest.
[0052] The term "halogen" as used herein refers to the substituents
fluoro, bromo, chloro, and iodo.
[0053] The term "poly-histidine affinity tag" as used herein refers
to a non-natural consecutive sequence of histidine amino acid
residues including any corresponding peptides disclosed in U.S.
Pat. Nos. 5,284,933 and 5,310,663. Typically such sequences
comprise four to ten histidine residues that are typically linked
to the carboxy and/or amino terminal end of a protein of interest.
Optionally, the poly-histidine affinity tag may be linked,
in-frame, in the middle of the protein of interest.
[0054] The term "indirect binding" as used herein refers to the
binding of the fluorescent compound to the affinity tag due to a
third component, typically a polyvalent metal ion. The fluorescent
compound and the affinity tag form a ternary complex with a metal
ion wherein the metal ion binds both the affinity tag and the
acetic acid groups of the fluorescent compound. The metal ion has
affinity for both the binding domain and affinity tag and as such
confers affinity to the binding domain for the affinity tag that
would not be present without the metal ion. Alternatively, a metal
ion has affinity for the binding domain that when bound induces a
conformational change that confers affinity to the binding domain
for the affinity tag. Thus, in this instance, the metal ion may not
have affinity for the affinity tag; however, the metal ion will
induce the binding domain to have affinity for the affinity
tag.
[0055] The term "indole" or "indole derivative" as used herein
refers to a compound generally having the formula and derivatives
thereof. 5
[0056] The fluorophore is substituted by a linker at any of the
aromatic carbon atoms and the linker attaches the fluorophore to a
binding domain. The fluorophore may also be further substituted by
substituents that adjust the solubility, spectral properties or
other physical properties of the fluorophore.
[0057] The term "isolated," as used herein refers to, a preparation
of peptide, protein or protein complex that is essentially free
from contaminating proteins that normally would be present in
association with the peptide, protein or complex, e.g., in a
cellular mixture or milieu in which the protein or complex is found
endogenously. In addition "isolated" also refers to the further
separation from reagents used to isolate the peptide, protein or
complex from cellular mixture. Thus, an isolated fusion protein may
be isolated from cellular components and optionally from the
fluorescent compounds of the present invention that normally would
contaminate or interfere with the study of the complex in
isolation, for example while screening for modulators thereof.
[0058] The term "kit" as used refers to a packaged set of related
components, typically one or more compounds or compositions.
[0059] The term "Linker" or "L" as used herein refers to a single
covalent bond or a series of stable covalent bonds incorporating
1-30 nonhydrogen atoms selected from the group consisting of C, N,
O, S and P that covalently attach the fluorophore to the binding
domain of the fluorescent compounds.
[0060] The term "metal chelator" or "metal chelating moiety" as
used herein refers to a chemical compound that combines with a
metal ion to form a chelate structure.
[0061] The term "metal ion" as used herein refers to any metal ion
that has an affinity for an affinity tag and/or a binding domain
and that can be used to indirectly complex the fluorescent compound
and the fusion protein together. Such metal ions include, but are
not limited to, Ni.sup.2+, Co.sup.2+, Zn.sup.2+, Cu.sup.2+,
Al.sup.3+, Ca.sup.2+, Ac.sup.3+, Fe.sup.3+ and Ga.sup.3+.
[0062] The term "NTA" as used herein refers to the metal chelating
group N.alpha.,N.alpha.-bis(carboxymethyl)-lysine and derivatives
thereof. Such derivatives include nitriloacetic acid.
[0063] The term "poly-arginine affinity tag" as used herein refers
to a consecutive sequence, typically 4-6, of arginine residues
(Nock et al (1997) FEBS Lett. 414(2):233-238).
[0064] The terms "protein" and "polypeptide" are used herein in a
generic sense to include polymers of amino acid residues of any
length. The term "peptide" is used herein to refer to polypeptides
having less than 250 amino acid residues, typically less than 100
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residues are an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers.
[0065] The term "protein of interest" as used herein refers to any
protein to which an affinity tag is fused to for the purpose of
detection, isolation, labeling, tagging, monitoring and
purification.
[0066] The term "sample" as used herein refers to any material that
may contain fusion proteins, as defined above. Typically, the
sample comprises endogenous host cell proteins. The sample may be
in an aqueous solution, a viable cell culture or immobilized on a
solid or semi solid surface such as a polymeric gel, polymeric
bead, membrane blot or on a microarray.
II. Compositions and Methods of Use
[0067] In accordance with the present invention, methods and
compositions are provided that label and detect fusion proteins by
specifically and selectively binding to an affinity tag of a fusion
protein. The affinity tag is defined to include any affinity tag
known to one skilled in the art and fused to a protein of interest
for the purposes of detection and purification. The fluorescent
compound is defined as being capable of binding to an affinity tag
and includes the general formula A(B)n wherein A is any fluorophore
known to one skilled in the art, B is a selected binding domain of
the present invention and n is an integer from 1 to 6. The binding
domain is a chemical moiety, protein or fragment thereof with the
proviso that the fluorescent compound does not comprise an antibody
or fragment thereof. The binding domain may interact directly,
selectively binding to the affinity tag, or indirectly, wherein a
third component forms a ternary complex between the fluorescent
compound and the affinity tag. Typically, the third component is a
metal ion wherein the metal ion has affinity for both the affinity
tag and the binding domain. Alternatively, the third component does
not have an affinity for the affinity tag but induces a
conformational change to the binding domain such that the binding
domain has an affinity for an affinity tag. The binding moiety can
be a charged chemical moiety such as a metal chelating group, a
protein, a peptide or fragment thereof such as calmodulin, provided
that the binding domain is not an antibody or generated from an
antibody. Thus, the present invention contemplates a wide range of
fluorescent compounds that can be used to detect a myriad of
affinity tags that are fused to a protein of interest whereby
detection of a fusion protein is determined by a fluorescent signal
generated from the fluorescent compound.
[0068] In addition to the components of the fluorescent compound
that confer selectivity for an affinity tag, the staining solution
also plays a critical role in determining selectivity and is
typically altered depending on the affinity tag and the assay
method. The staining solution contains a buffer and a fluorescent
compound wherein the buffering components fine-tune the selectivity
of the fluorescent compound for an affinity tag. For example, we
have found that for the selective detection of poly-histidine
containing fusion proteins on a gel that the buffer is preferably
slightly acidic or neutral, contains a salt and has a pKa of about
6.0 to about 7.05. It appears that a pKa value of the buffer that
is similar to the pKa value of the imidazole ring of histidine,
which is about 7.05, results in a buffer that facilitates the
non-covalent binding of the fluorescent compound to the
poly-histidine affinity tag. Thus, preferred buffers for the
detection of poly-histidine affinity tag containing fusion proteins
includes, but are not limited to, Good's buffer, PIPES and MOPS
buffers.
[0069] A. Components of the Fluorescent Compounds
[0070] The present invention provides fluorescent compounds that
have an affinity for a number of affinity tags. When the binding
domain is a protein, typically there is a short linker, less than
10 nonhydrogen atoms that covalently attach the fluorophore to the
protein-binding domain. The protein-binding domain may interact
directly or indirectly through a metal ion with the affinity tag.
When the binding domain is a charged chemical moiety the
fluorescent compounds of the present invention have the general
formula A(L)m(B) n wherein A is a fluorophore, L is a Linker, B is
a binding domain, m is an integer from 1 to 4 and n is an integer
from 1 to 6. By selection of an appropriate binding domain, a
corresponding affinity tag can be selectively and non-covalently
labeled with a fluorophore. The fluorophore typically has a passive
role in the affinity of the binding domain for the affinity tag,
although the fluorophore may be substituted to alter the affinity
of the covalently attached binding domain. However, we have found
that fluorophores that are substituted by sulfonated groups tend to
reduce the selectivity of the fluorescent compound for the affinity
tag. Therefore, one skilled in the art will appreciate that any
fluorophore, or derivative thereof, can be covalently linked using
an appropriate Linker(s) to a specific binding domain resulting in
a significant advancement in the ability to fluorescently detect
fusion proteins that contain an affinity tag.
[0071] 1. Fluorophores of the Fluorescent Compounds
[0072] A fluorophore of the present invention is any chemical
moiety that exhibits an absorption maximum beyond 280 nm, and when
covalently linked to a binding domain of the present invention
forms a fluorescent compound. The covalent linkage can be a single
covalent bond or a combination of stable chemical bonds. The
covalent linkage attaching the fluorophore to the binding domain is
typically a substituted alkyl chain that incorporates 1-30
nonhydrogen atoms selected from the group consisting of C, N, O, S
and P. Optionally, the linker can be a single covalent bond or the
alkyl chain can incorporate a benzene ring.
[0073] Fluorophores of the present invention include, without
limitation; a pyrene, an anthracene, a naphthalene, an acridine, a
stilbene, an indole or benzindole, an oxazole or benzoxazole, a
thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole
(NBD), a cyanine (including any corresponding compounds in U.S.
Pat. No. 5,863,753), a carbocyanine (including any corresponding
compounds in U.S. Ser. Nos. 09/557,275; 09/968,401 and 09/969,853
and U.S. Pat. Nos. 6,403,807; 6,348,599; 5,486,616; 5,268,486;
5,569,587; 5,569,766; 5,627,027 and 6,048,982), a carbostyryl, a
porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a
pyridine, a quinoline, a borapolyazaindacene (including any
corresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;
5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene
(including any corresponding compounds disclosed in U.S. Pat. Nos.
6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343, 5,798,276
and U.S. Ser. No. 09/922,333), an oxazine or a benzoxazine, a
carbazine (including any corresponding compounds disclosed in U.S.
Pat. No. 4,810,636), a phenalenone, a coumarin (including an
corresponding compounds disclosed in U.S. Pat. Nos. 5,696,157;
5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an
corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and
4,849,362) and benzphenalenone (including any corresponding
compounds disclosed in U.S. Pat. No. 4,812,409) and derivatives
thereof. As used herein, oxazines include resorufins (including any
corresponding compounds disclosed in U.S. Pat. No. 5,242,805),
aminooxazinones, diaminooxazines, and their benzo-substituted
analogs.
[0074] Where the fluorophore is a xanthene, the fluorophore is
optionally a fluorescein, a rhodol (including any corresponding
compounds disclosed in U.S. Pat. Nos. 5,227,487 and 5,442,045), or
a rhodamine (including any corresponding compounds in U.S. Pat.
Nos. 5,798,276 and 5,846,737). As used herein, fluorescein includes
benzo- or dibenzofluoresceins, seminaphthofluoresceins, or
naphthofluoresceins. Similarly, as used herein rhodol includes
seminaphthorhodafluors (including any corresponding compounds
disclosed in U.S. Pat. No. 4,945,171).
[0075] Preferred fluorophores of the invention include xanthene,
coumarin, cyanine, acridine, anthracene, benzofuran, indole and
borapolyazaindacene. Most preferred are cyanine,
borapolyazaindacene, coumarin and benzofuran. The choice of the
fluorophore attached to the binding domain will determine the
fluorescent compound's absorption and fluorescence emission
properties. It is an aspect of the present invention that the
fluorophore not be sulfonated.
[0076] 2 . . . Linkers of the Fluorescent Compounds
[0077] As described above, the fluorophores of the present
invention are covalently attached to a binding domain by a Linker
to form the fluorescent compounds of the present invention. The
Linker typically incorporates 1-30 nonhydrogen atoms selected from
the group consisting of C, N, O, S and P. The linker is typically a
substituted alkyl or a substituted cycloalkyl. Alternately, the
fluorophore may be directly attached (where Linker is a single
bond) to the binding domain or the alkyl may contain a benzene
ring. When the linker is not a single covalent bond, the linker may
be any combination of stable chemical bonds, optionally including,
single, double, triple or aromatic carbon-carbon bonds, as well as
carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen
bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen
bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds.
Typically the linker incorporates less than 20 nonhydrogen atoms
and are composed of any combination of ether, thioether, urea,
thiourea, amine, ester, carboxamide, sulfonamide, hydrazide bonds
and aromatic or heteroaromatic bonds. Typically the linker is a
combination of single carbon-carbon bonds and carboxamide,
sulfonamide or thioether bonds. The bonds of the Linker typically
result in the following moieties that can be found in the Linker:
ether, thioether, carboxamide, thiourea, sulfonamide, urea,
urethane, hydrazine, alkyl, aryl, heteroaryl, alkoky, cycloalkyl
and amine moieties. Examples of typical fluorescent compounds
incorporate the following three (V, VI and VII) Linker formulas:
Formula (V)
--(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.-
d--, Formula (VI)
--((C.sub.6R".sub.4)O).sub.d(CH.sub.2).sub.e(C(X)NH(CH.s-
ub.2).sub.e)(NH).sub.dC(X)NH(C.sub.6R.sub.14)(CH.sub.2).sub.e-- and
Formula (VII) --(O).sub.d(CH.sub.2).sub.tO(C.sub.6R".sub.4)--
wherein X is O or S, d is 0-1, e is 1-6, f is 2 or 3, and R" is
independently H, halogen, alkoxy or alkyl. It is understood that X,
d, e and are independently selected within a linker.
[0078] Thus, a selected embodiment of the present invention is the
following fluorescent compound formulas (VII, IX and X): Formula
(VIII)
(A)-[(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d-
].sub.m--(B).sub.n; Formula (IX)
(A)-((C.sub.6R".sub.4)O).sub.d(CH.sub.2).-
sub.e(C(X)NH(CH.sub.2).sub.e)(NH).sub.dC(X)NH(C.sub.6R.sub.14)(CH.sub.2).s-
ub.e--(B) and Formula (X)
(A)-(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4)-- -(B) wherein m is
an integer from 1 to 4 .mu.m is an integer from 1 to 6, A is a
fluorophore and B is a binding domain. Particularly preferred is
Formula (VIII) wherein d is 0, e is 1 to 4, X is O, m is 2 and n is
2 or Formula (VIII) wherein d is 1 and e is 1 or 2. Preferred
embodiments of Formula (X) is when d is 0, f is 2, or a variation
of Formula (X) having the Formula (XI)
(B)(L)(A)--(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4)--- (B)
wherein L is a single covalent bond, B is a binding domain, A is
fluorophore, d is 1 and f is 2.
[0079] Any combination of linkers may be used to attach the
fluorophore and the binding domain together, typically a
fluorophore will have one or two linkers attached that may be the
same or different. In addition, a linker may have more than one
binding domain per linker. The linker may also be substituted to
alter the physical properties of the fluorescent compound, such as
binding affinity of the binding domain and spectral properties of
the fluorophore. For fluorescent compounds that have an affinity
for the poly-histidine affinity tag, the linker typically
incorporates an oxygen atom due to its ability to increase the
affinity of the acetic acid binding domain, described below, for
the affinity tag. This feature of the linker is especially true for
fluorescent compound Formula (XI)
(B)(L)(A)--(O).sub.d(CH.sub.2).sub.fO(C.sub.6R'.sub.4)--(B). Thus,
an important feature of the linker is to alter the binding affinity
of the binding domain by increasing the affinity with the
incorporation of oxygen into the linker.
[0080] The linker can also have other substituents that alter the
binding affinity of the binding domain. The benzene ring
(C.sub.6R".sub.4) of Formula (XI) is typically substituted with a
halogen, preferably chlorine or fluorine, which tune the affinity
of the binding domain. These halogen substituents appear to lower
the affinity of binding domain but increase the specificity of the
binding domain for the affinity tag resulting in overall increased
sensitivity of the fluorescent compound for the affinity tag. Thus,
linker substituents function to tune the binding affinity of the
fluorescent compound to optimize the sensitivity of the binding
domain for the affinity tag
[0081] Another important feature of the linker is to provide an
adequate space between the fluorophore and the binding domain so as
to prevent the fluorophore from providing a steric hindrance to the
binding of the affinity tag for the binding domain of the
fluorescent compound. Thus, when a binding domain is attached to
the fluorophore by a single covalent bond there is typically
another linker containing an oxygen atom attached to the same
fluorophore at a different position to increase the affinity of
both binding domains for the affinity tag. Therefore, the linker of
the present fluorescent compounds is important for (1) coupling the
fluorophore to the binding domain, (2) providing an adequate space
between the fluorophore and the binding domain so as not to
sterically hinder the affinity of the binding domain and the
affinity tag and (3) altering the affinity of the binding domain
for the affinity tag either by the choice of the atoms of the
linker or indirectly by addition of substituents to the linker.
[0082] 3. Binding Domains of the Fluorescent Compounds
[0083] The binding domain of the present fluorescent compounds,
include without limitation, charged chemical moieties, a proteins
or fragments thereof that are capable of non-covalently binding to
an affinity tag of the present invention. The binding domain,
either independently or when complexed with a metal ion, has
specific and selective affinity for an affinity tag containing
fusion protein. The fluorescent compounds, A(L)m(B)n, may have more
than one linker and more than one binding domain, which may or may
not be the same. Preferably, the binding domains are all selective
for the same affinity tag, however for certain applications it may
be desirable to have one fluorophore linked to binding domains that
have selective affinity for different affinity tags. In this
manner, selection and orientation of the binding domain relative to
the fluorophore is critical for the specificity, sensitivity and
selectivity of the binding domain.
[0084] The present invention contemplates protein and
peptide-binding domains that are not antibodies or fragments
thereof. Thus, an aspect of the present invention is affinity tags
that are selective for such proteins, and these include without
limitation, GST, calmodulin, maltose-binding, and chitin-binding
affinity tags. These peptides bind the glutathione tripeptide,
calmodulin protein, maltose and chitin respectively. When these
polypeptides are attached by a linker to a fluorophore, they
function to site-specifically label these affinity tag containing
fusion proteins.
[0085] Calmodulin selectively and with high affinity binds calcium
ions, the calcium ions then induce a conformation change that
causes the protein to have affinity for the calmodulin affinity tag
(Hentz N G et al. (1996) Anal Chem 68:1550-5; Zheng C F et al.
(1997) 186:55-60). A fluorophore of the present invention that is
covalently attached to calmodulin effectively attaches the
fluorophore to the calmodulin affinity tag and subsequently a
protein of interest. Thus, a staining solution specific for
calmodulin affinity tag containing fusion proteins would include,
at a minimum, a fluorescent compound comprising calmodulin and
calcium ions.
[0086] In contrast, the glutathione tripeptide binds directly to
the GST affinity tag (Kaplan W et al (1997) Protein Sci. 6:399406;
Lew A M et al (1991) J. Immunol. Methods 136:211-9). A fluorescent
compound covalently attached to glutathione effectively attaches a
fluorophore to a GST affinity tag containing fusion protein. In
this way, fluorescent compounds comprising glutathione, provide an
effective means for detecting such fusion proteins in a gel or
solution, a means not previously feasible with currently known
compounds, See Example 20. Thus, an aspect of the invention is
detection of GST affinity tag containing fusion proteins with a
fluorescent compound that comprises the tripeptide glutathione.
Preferred fluorescent compounds comprise a xanthene
fluorophore.
[0087] An important aspect of the present invention includes
charged chemical moieties that have affinity for an affinity
peptide. These moieties include, without limitation, acetic acid
groups, phosphates and sulfates. Particularly preferred are binding
domains that have affinity for positively charged affinity tags
such as poly-histidine or poly-arginine affinity tag containing
fusion proteins. These binding domains typically contain terminal
acetic acid groups. The acetic acid binding domains contain
nitrogen as the point of attachment for the acetic acid groups and
the binding domain is attached to a linker at either a nitrogen or
carbon atom depending on one of the three (I, II or III) formulas
for the binding domain. Specifically, the acetic acid binding
domains have formula (I) .sup.-O.sub.2CCH(R)N(CH.sub.2CO.sup.-.su-
b.2).sub.2, wherein R is a linker that is covalently bonded to the
methine carbon atom (See, for example Compound 1), or formula (II)
--N(CH.sub.2CO.sub.2).sub.2 wherein the linker is covalently bonded
to a nitrogen atom (See, for example Compound 12). Alternatively,
the acetic acid binding domain has formula (III)
(CH.sub.2Co.sup.-.sub.2).sub.ZN[(CH-
(R)).sub.SN(CH.sub.2CO.sup.-.sub.2)].sub.T(CH(R)).sub.SN(CH.sub.2CO.sup.-.-
sub.2).sub.Z wherein the linker is attached to a methine carbon or
nitrogen atom and Z is 1 or 2, S is 1 to 5 and T is 0 to 4. In all
cases, the acetic acid binding domain contains at least two acetic
acid groups and a nitrogen atom is the point of attachment for the
acetic acid groups. When a binding domain that contains only two
acetic acid groups is attached to a fluorophore either (1) another
acetic acid binding domain is also attached to the fluorophore or
(2) an acetic acid group is attached by a linker to the
fluorophore. This is because the fluorescent compounds with at
least three acetic acid groups is preferable for providing
selective and sensitive affinity for the poly-histidine affinity
tag.
[0088] The acetic acid binding domains are typically part of a
metal chelating moiety such as BAPTA, IDA, NTA, DTPA and TTHA.
BAPTA, as used herein, refers to analogs, including derivatives, of
the metal chelating moiety
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and
salts thereof including any corresponding compounds disclosed in
U.S. Pat. Nos. 4,603,209; 4,849,362; 5,049,673; 5,453,517;
5,459,276; 5,516,911; 5,501,980; and 5,773,227. IDA, as used
herein, refers to imidodiacetic acid compounds and derivatives
thereof. DTPA, as used herein, refers to diethylenetriamine
pentaacetic acid compounds and derivatives thereof including any
corresponding compounds disclosed in U.S. Pat. Nos. 4,978,763 and
4,647,447. NTA, as used herein, refers to Na,
Na-bis(carboxymethyl)-lysine and derivatives thereof, such
derivatives including nitriloacetic acid. TTHA, as used herein,
refers to triethylenetetramine hexaacetic acid and derivatives
thereof.
[0089] The acetic acid binding domain may comprise the entire metal
chelating moiety or only be part of such a moiety. A binding domain
that encompasses an entire chelating moiety is represented by the
formulas (I) .sup.--O.sub.2CCH(R)N(CH.sub.2CO.sup.-.sub.2).sub.2,
and (III)
(CH.sub.2CO.sup.-.sub.2).sub.ZN[(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2)].s-
ub.T(CH(R)).sub.SN(CH.sub.2CO.sup.-.sub.2).sub.Z wherein these
formulas comprise the chelating moieties NTA (Formula I), DTPA and
TTHA (Formula II). The binding domain having the formula (II)
N(CH.sub.2CO.sub.2).sub.2 comprises, in part, the chelating
moieties IDA and BAPTA. When a binding domain is only part of a
chelating moiety such as BAPTA, the remaining part of the chelating
moiety comprises the linker of a fluorescent compound or the
fluorophore. This is demonstrated by the fluorescent compound
Formula (X) (A)--(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4)--(B-
), wherein the represented linker is part of the BAPTA chelating
moiety. The remaining phenyl ring of the BAPTA moiety, when
present, is typically part of the fluorophore, as demonstrated by
Compound 12.
[0090] Due to the inclusion of chelating moieties in the binding
domain and/or linker of the fluorescent compounds these moieties
can be optionally substituted to adjust the binding affinity,
solubility, or other physical properties of the compound. This is
particularly true for the BAPTA chelating moiety wherein the
benzene ring (C.sub.6R".sub.4) of the linker Formula (VII)
--(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4)-- is optionally
substituted. Particularly advantageous substitutions are halogen
substituents, especially fluorine and chlorine. Without wishing to
be bound by a theory, it appears that these substituents, as
electron withdrawing groups, tune the affinity of the binding
domain for the affinity tag or a metal ion resulting in increased
stability of the complex.
[0091] In addition, because the acetic acid binding domain contains
all or part of a number of chelating moieties these binding domains
also have affinity for metal ions. This aspect of the binding
domain is useful for certain fluorescent compounds. However we have
unexpectedly discovered that nickel ions are not necessary for the
detection of poly-histidine affinity tag containing fusion proteins
(FIG. 1). While this is an important aspect of the present
invention, for some compounds, inclusion of the metal ion in a
staining solution may be desirable. This is because for certain
compounds, inclusion of metal ions into a staining solution may
stabilize the complex of the fluorescent compound and the affinity
tag containing fusion protein. Thus, for certain compounds, the
inclusion of a metal ion is beneficial. Alternatively, as
demonstrated in FIGS. 1 and 2, the acetic acid binding domain has
selective affinity for the poly-histidine affinity tag due to the
negative charge of the acetic acid groups and the positive charge
of the poly-histidine affinity tag at a neutral or mildly acidic
pH, and in fact, as FIG. 1 demonstrates an increase in signal
intensity is obtained when the staining solution does not contain
nickel ions.
[0092] B. Combination of Components to Form Fluorescent
Compounds
[0093] The components of the fluorescent compound having now been
described, combination of certain fluorophores, linkers and binding
domains are provided to demonstrate the complexity of the
fluorescent compounds and their application. While it has been
stressed that a wide range of components can be used to make the
fluorescent compounds it should also be understood that the
individual selection of components to make a particularly useful
fluorescent compound requires an understanding of the fluorophores,
the linkers, the binding domain and how certain combinations
function to selectively bind to affinity tags. Therefore, what
follows are selected fluorophores indicating sites of attachment,
substituents and preferred linkers along with binding domains.
However, the following description is in no way limiting and should
not be construed as the only preferred embodiments as many
fluorophores with linkers attached are equally as preferred. It is
understood that the following compounds comprise the salts, acids
and lipophilic forms including esters of the compounds, as
particular forms are advantageous in certain applications.
Compounds that comprise acetyloxy methyl (AM) ester are
particularly useful for intracellular labeling of affinity tag
containing fusion proteins wherein fluorescent compounds comprising
AM ester moieties easily enter cells where the ester is cleaved
resulting in terminal acetic acid groups on the fluorescent
compound. In this way, newly translated fusion proteins can be
detected, in vivo, and monitored to ascertain information about the
functional proteome of the cell including discovery of drug
targets. The terminal acetic acid groups are typically part of the
binding domain but they may be other places on the compound.
[0094] The linkers of the present invention can be attached at many
positions on the fluorophore resulting in an exponential number of
fluorescent compounds contemplated by the present invention.
Preferred fluorophores of the fluorescent compounds are cyanine,
coumarin, borapolyazaindacene, benzofuran and xanthenes including
rhodol, rhodamine, fluorescein and derivatives thereof.
[0095] Most preferred fluorophores of the fluorescent compounds are
coumarin and borapolyazaindacene. The coumarin fluorophore has the
Formula (XII), as shown below, wherein A is NH.sub.2, OR' or
N(R').sub.2, R' is H, an alkyl or an acetic acid binding domain and
R.sup.9-R.sup.12 and R.sup.8 can be any of the corresponding
substituents disclosed in U.S. Pat. Nos. 5,696,157 and 5,830,912,
supra. Typical substituents include halogen, lower alkyl, alkoxy
and hydrogen. 6
[0096] Particularly preferred fluorescent compounds with coumarin
as a fluorophore are exemplified in compounds 1, 4, 5 and 6. These
exemplified compounds comprise a linker at R.sup.9 or R.sup.10
having the formula
--(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d--
wherein R.sup.9 or R.sup.10 that is not a linker is typically a
methyl group, R.sup.11 is typically hydrogen, R.sup.12 is fluorine
(compound 4), sulfonic acid (compound 1) or hydrogen (compound 5
and 6). R.sup.12 is typically hydrogen, however a preferred
substituent is fluorine (compound 4). Thus, a preferred compound of
the present invention has Formula (VIII)
(A)-[(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e-
).sub.d].sub.m--(B).sub.n wherein A is a coumarin, B is an acetic
acid binding domain and d of the linker is typically 0.
[0097] Alternatively, the coumarin of Formula (XII) can be any of
the compounds disclosed in U.S. Pat. Nos. 5,459,276 and 5,501,980.
These compounds comprise a linker at R.sup.12 and the fluorescent
compound Formula (XI)
(B)(L)(A)-(O).sub.d(CH.sub.2).sub.fO(C.sub.6R.sub.14)--(B) wherein
(B)(L) is A of fluorophore Formula (XII).
[0098] It is understood that the linkers of the present invention
may be attached at any of R.sup.8-R.sup.12, and that any of the
binding domains of the present invention can be attached to the
linker.
[0099] The borapolyazaindacene fluorophore has the formula (XIII),
as shown below, wherein R.sup.1-R.sup.7 can be substituted by any
of the corresponding substituents disclosed in U.S. Pat. Nos.
5,187,288; 5,248,782 and 5,274,113, supra. Typical substituents
include heteroaryl, aryl, lower alky, alkoxy and hydrogen. 7
[0100] Particularly preferred fluorescent compounds with
borapolyazaindacene as a fluorophore are exemplified in compounds
2, 3 and 7-14. These exemplified compounds comprise a linker at
R.sup.7, Re, R.sup.2 or R.sup.1 having the Formula (VI)
--((C.sub.6R'.sub.4)O).sub.d(C-
H.sub.2).sub.e(C(X)NH(CH.sub.2).sub.e)(NH).sub.dC(X)NH(C.sub.6R'.sub.4)(CH-
.sub.2).sub.e-- and/or Formula (V)
--(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.-
e(NHC(X)(CH.sub.2).sub.e).sub.d--, wherein the fluorophore is
attached by one linker or two linkers. When the fluorophore is
attached by two linkers, the linkers are typically present at
R.sup.6 and R.sup.2 (Compound 3) or R.sup.7 and R.sup.1 (Compound
2) and further attached to an acetic acid binding domain. Thus,
preferred fluorescent compounds of the present invention have the
formula Formula (VIII)
(A)-[(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d-
].sub.m--(B).sub.n; Formula (IX)
(A)-((C.sub.6R'.sub.4)O).sub.d(CH.sub.2).-
sub.e(C(X)NH(CH.sub.2).sub.e)(NH).sub.dC(X)NH(C.sub.6R'.sub.4)(CH.sub.2).s-
ub.e(B), wherein A is borapolyazaindacene and B is an acetic acid
binding domain having Formula (I)
.sup.-O.sub.2CCH(R)N(CH.sub.2CO.sup.-.sub.2).su- b.2, or formula
(III) (CH.sub.2CO.sup.-.sub.2).sub.ZN[(CH.sub.2).sub.SN(CH-
.sub.2CO.sup.-.sub.2)].sub.R(CH.sub.2).sub.SN(CH.sub.2CO.sup.-.sub.2).sub.-
Z.
[0101] Fluorescent compounds comprising acetic acid binding domain
Formula (III) can also be used to calorimetrically detect
poly-histidine affinity tag containing fusion proteins with the
same sensitivity as the fluorescent signal.
[0102] The linker and non-linker substituents of the
borapolyazaindacene can be present at any of R.sup.1-R.sup.7. The
linkers may be the same or different and may be attached to the
same or different binding domains. In this way a fluorescent
compound may have affinity for one or more different affinity
tags.
[0103] The benzofuran fluorophore has the Formula (XIV), as shown
below, wherein R.sup.13-R.sup.18 can be any of the corresponding
substituents disclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362,
supra. Typical non-linker substituents include hydrogen and
substituted heteroaryl. 8
[0104] Typical fluorescent compounds comprising a benzofuran
fluorophore contain a linker attached at R.sup.14 and R.sup.15, the
compounds typically comprise two linkers, one of which is a single
covalent bond at R.sup.14 and a linker attached at the R.sup.15
position comprising Linker Formula (VII)
--(O).sub.d(CH.sub.2).sub.fO(C.sub.6R".sub.4)--, such a compound is
demonstrated in Compound 12. 9
[0105] R.sup.18 is typically substituted by a substituted
heteroaryl, as Compound 12 demonstrates, preferably an oxazole.
Compound 12 also demonstrates a substitution on the benzene ring
(C.sub.6R.sub.14) of the linker; typically the ring is substituted
with a halogen, preferably fluorine or chlorine.
[0106] The xanthene fluorophore has the Formula (XV), as shown
below, wherein A, B and R.sup.19-R.sup.25 can be any of the
corresponding substituents disclosed in U.S. Pat. Nos. 6,162,931;
6,130,101; 6,229,055; 6,339,392 and 5,451,343, supra. Typically, A
is NR.sub.12 or OR', B is OR' or NR.sub.2, wherein R' is hydrogen,
an alkyl group or an acetic acid binding domain. 10
[0107] The linkers of the present invention can be present at any
of the R groups and with any of the binding domains of the present
invention.
[0108] When R.sup.25 is substituted with a benzene ring
(C.sub.6R".sub.4), as shown below for Formula (XVI), the
fluorophore is a rhodol, a rhodamine or a fluorescein depending on
A and B. Rhodol fluorophores are represented when A is NH.sub.2 and
B is O, rhodamine fluorophores are represented when A is NH.sub.2
and B is NH.sub.2 and fluorescein fluorophores are represented when
A is OH and B is O. These fluorohpores can be substituted by any of
the corresponding substituents disclosed in U.S. Pat. Nos.
5,227,487; 5,442,045; 5,798,276; 5,846,737; 6,162,931; 6,130,101;
6,229,055; 6,339,392 and 5,451,343, supra. 11
[0109] The cyanine fluorophore has the Formula (XVII), as seen
below, wherein R.sup.31-R.sup.40 and R.sup.31-R.sup.40' can be
substituted by any of the corresponding substtuents disclosed in
the U.S. Ser. Nos. 09/968/401 and 09/969,853 and U.S. Pat. Nos.
6,403,807; 6,348,599; 5,486,616; 5,268,486; 5,569,587; 5,569,766;
5,627,027 and 6,048,982, supra. In addition the linkers of the
present invention can be substituted at any of the R groups,
preferably R.sup.40, R.sup.31, R.sup.40, R.sup.31', R.sup.39 and
R.sup.39, and subsequently attached by a binding domain of the
present invention. 12
[0110] C. Methods of Use
[0111] The fluorescent compounds of the present invention may be
utilized without limit for the site-specific labeling of affinity
tags that results in detection of a fusion protein containing a
protein of interest and an affinity tag. The methods for detecting
a fusion protein containing an affinity tag include contacting a
sample with a staining solution and then illuminating the sample
whereby the fusion protein is detected. The staining solution
comprises 1) an appropriate fluorescent compound that is capable of
selectively binding, directly or indirectly, to an affinity tag and
2) a buffer.
[0112] Typically, the staining solution comprises a fluorescent
compound capable of binding to poly-histidine, poly-arginine, and
GST affinity tags wherein the binding domain is selected from the
group consisting of glutathione, a positively charged chemical
moiety and a negatively charged chemical moiety including acetic
acid groups. The fluorophore is selected from the group consisting
of xanthene, coumarin, cyanine, acridine, anthracene, benzofuran,
indole and borapolyazaindacene. In one aspect of the present
invention the staining solution for detecting fusion proteins
comprising poly-histidine or poly-arginine affinity tags
comprises:
[0113] 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,
[0114] b) a buffer having a pH about 5 to 6.9 and comprising an
acceptable counter ion.
[0115] In addition, we have found that for the selective detection
of poly-histidine affinity tag containing fusion proteins that the
buffer preferraly contains a salt and has a pKa of about 6.0 to
about 7.5. Thus, preferable buffers for this application include
Good's buffer, MOPS and PIPES buffers.
[0116] An example of an appropriate matching of a fluorescent
compound and affinity tag is a poly-histidine affinity tag and a
fluorescent compound that contains an acetic acid binding domain.
The acetic acid binding domain is capable of selectively
interacting with either a metal ion or the positively charged
poly-histidine affinity tag.
[0117] In one aspect of the invention specific fluorescent
compounds are used to detect and label fusion proteins that contain
a poly-histidine affinity tag. A method of the present invention
wherein the poly-histidine affinity tag containing fusion protein
is detected after being separated on a polyacrylamide gel comprises
the following steps:
[0118] i) immobilizing said sample on a solid or semi-solid
matrix;
[0119] ii) optionally contacting said sample of step i) with a
fixing solution;
[0120] iii) contacting said sample of step 1) or ii) with a
staining solution comprising a buffer having a pH about 5 to 6.9
and 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;
[0121] iv) incubating said staining solution and said sample for
sufficient time to allow said compound to associate either directly
or indirectly with said poly-histidine affinity tag;
[0122] v) illuminating said fluorophore with a suitable light
source whereby said fusion protein is detected.
[0123] In step one (1) a sample, obtained as described below, is
prepared in an appropriate buffer and immobilized on a solid or
semi-solid matrix. Typically the sample is separated on a gel,
typically a SDS-polyacrylamide gel. Alternatively, the sample is
immobilized on solid or semi-solid matrix that includes a membrane,
polymeric beads, polymeric gel, a glass surface or an array
surface. When SDS-polyacrylamide gels are employed, the denaturing
effects of the SDS buffer allow for the exposure of the affinity
tag because when folded into a native form the affinity tag can be
obscured from compounds that have affinity for the peptide. Thus,
SDS gel electrophoresis facilitates the binding of the fluorescent
compounds of the present invention with the poly-histidine affinity
tag of a fusion protein. However, after the sample has been
separated it is important that the SDS be removed from the gel with
a fixing solution for maxima detection of the affinity tag because
the SDS interferes with the affinity of the fluorescent compound
for the affinity tag.
[0124] Therefore, the second (2) step optionally comprises
incubating the gel in a fixing solution that typically includes an
alcohol so as to remove the SDS before the staining solution is
added to the gel. Typically, effective removal of SDS requires a
step-wise contact with the fixing solution wherein the fixing
solution is incubated with the gel, removed and new solution is
added for an additional time period. Following the fixing step, the
gel is typically rinsed with water.
[0125] During the third (3) and fourth (4) steps, the gel
containing the sample, is contacted with a staining solution for a
time period that permits effective non-covalent labeling of the
fluorescent compound to the affinity tag. Typically this time
period is from about 30 minutes to about 120 minutes. The staining
solution contains a fluorescent compound that is capable of
directly or indirectly binding to the affinity tag of the fusion
protein and has the general formula A(L)m(B)n, as described above.
For the binding of poly-histidine affinity tags, fluorescent
compounds that contain an acetic acid-binding domain are preferred.
Exemplified compounds 1-16 are particularly preferred. The staining
solution optionally comprises an appropriate metal ion, an
appropriate metal ion being one that has affinity for both the
fluorescent compound and the affinity tag. As described above, some
of the affinity tags have an affinity for metal ions, therefore for
particular applications; a metal ion is desirable in the staining
solution. The staining solution may be pre-mixed and added to the
gel in one step or the individual components may be added step-wise
to the gel. Preferably the gel is subjected to mild agitation while
in contact with the staining solution.
[0126] During the fifth (5) step, the gel is illuminated with a
suitable light source that allows for the fluorophore of the
fluorescent compound affinity tag complex to be visualized whereby
the fusion proteins containing a poly-histidine affinity tag is
detected. Preferably, the gel is rinsed with water to remove
unbound fluorescent compound prior to illumination. The suitable
light source is dictated by the fluorophore of the fluorescent
compound. For example, a staining solution comprising compound 1
exhibits bright-blue fluorescence (emission maximum=450 nm) when
illuminated with UV-A or UV-B light from a standard ultraviolet
transilluminator and compound 2 exhibits bright-green fluorescence
(emission maximum=515 nm) when illuminated with visible light from
a laser-based gel scanner equipped with a 470 nm second-harmonic
generation (SHG) or 488 nm argon ion laser source. Typically,
detection limits of poly-histidine affinity tag containing fusion
proteins using staining solution containing Compound 1 or 2 is 2565
ng in whole cell lysates.
[0127] In another aspect, the invention includes a complex of a
fluorescent compound that is capable of selectively binding,
directly or indirectly, to an affinity tag containing fusion
protein and a fusion protein. This complex may be a ternary complex
wherein a third component such as a metal ion has affinity for both
the affinity tag and the fluorescent compound. Alternatively, the
complex may be present without the metal ion.
[0128] 1. Sample Preparation
[0129] The fusion proteins of the invention can be expressed in a
number of systems including genetically engineered animals or
plants, or in cells such as bacteria, yeast, insect, plant and
mammalian cell cultures. The preparation of fusion proteins
comprising an affinity tag can be made using standard recombinant
DNA methods. Typically, a protein of interest, which is determined
by the end user, is synthesized and inserted into a vector
containing an affinity tag such that when inserted in frame the
affinity tag and protein of interest will be translated as one
fusion protein. There are many vectors that are available to one
skilled in the art that contain nucleotide sequence for an affinity
tag, such as pGEX (Amersham Biosciences) for GST affinity tag, pCAL
(Stratagene) for calmodulin affinity tag, pFLAG (Sigma Aldrich) for
FLAG affinity tag, 6.times.his tag vector (Qiagen) for
poly-histidine affinity tag and expression vectors for Glu-Glu
affinity tag including many expression systems available from
Invitrogen containing vectors with a combination of affinity tags
(U.S. Pat. No. 6,270,969). Alternatively, a nucleotide sequence
coding for a desired affinity tag is first synthesized and then
linked to a nucleotide sequence coding for the protein of interest.
This fused polynucleotide is then inserted into an expression
vector using techniques well known to those skilled in the art,
wherein the fusion protein will be expressed when the vector is
induced in a host cell such as E. coli. (Maniatis et al. "Molecular
Cloning" (2002), Cold Spring Harbor Laboratory).
[0130] Expression systems for expressing the fusion proteins are
available using E. coli, Bacillus sp. (Palva, I. et al., (1983)
Gene 22:229-235; Mosbach, K. et al., (1983) Nature 302:543-545)
Yeast and Salmonella. The polynucleotides encoding the fusion
proteins can also be ligated to various expression vectors for use
in transforming mammalian or insect cell cultures. Illustrative
examples of mammalian cell lines include VERO, COS, and HeLa cells,
Chinese hamster ovary (CHO) cell lines, and various cell lines
available from American Type Culture Collection (Bethesda, Md.).
Suitable insect cell lines include mosquito larvae, silkworm,
armyworm, moth and Drosophila cell lines.
[0131] Expression and isolation of fusion proteins are also well
known in the art (Maniatis et al, supra). Essentially, a suitable
host organism is transformed with an expression vector in which the
protein of interest or fused polynucleotide described above is
operably linked to an expression control sequence. The transformed
host cells are grown under suitable growth conditions wherein the
expression vector is induced to produce fusion proteins. When the
fusion protein is secreted out of the host organism the cell
culture media is collected and the soluble proteins are
concentrated. Alternatively, when the fusion protein is an
intracellular protein the host cells are pelleted and using
standard techniques the proteins are extracted wherein preferably
the DNA and lipids of the cell are removed from the crude cellular
extract.
[0132] When the sample is to be separated on a SDS-polyacrylamide
gel the sample is first equilibrated in an appropriate buffer, such
as a SDS-sample buffer containing Tris, glycerol, DTT, SDS, and
bromophenol blue.
[0133] Alternatively, the constructs encoding the fusion protein of
the invention are used to produce a genetically engineered animal
or plant. For production of genetically engineered animals (e.g.,
mice, rats, guinea pigs, rabbits, and the like) the construct can
be introduced into cells in vitro or in vivo. These nucleic acids
can be inserted into any of a number of well-known vectors for the
transfection of target cells and organisms.
[0134] After expression in the genetically engineered animal, the
fusion protein is detected in a sample from the animal. The sample
can be a biological fluid such as whole blood, plasma, serum, nasal
secretions, sputum, saliva, urine, sweat, transdermal exudates,
cerebrospinal fluid, or the like. Alternatively, the sample may be
whole organs, tissue or cells from the animal. Examples of sources
of such samples include muscle, eye, skin, gonads, lymph nodes,
heart, brain, lung, liver, kidney, spleen, solid tumors,
macrophages, mesothelium, and the like. In addition, the fusion
protein may be detected intracellularly wherein a live-cell version
of the present fluorescent compounds are used.
[0135] 2. Staining Solution
[0136] The staining solution can be prepared in a variety of ways,
which is dependent on the medium the sample is in. A particularly
preferred staining solution is one that is formulated for detection
of affinity tags in a gel. Specifically, the staining solution
comprises a fluorescent compound of the present invention in an
aqueous solution; optionally the staining solution comprises an
organic solvent and a buffering component. The selection of the
fluorescent compound dictates, in part, the other components of the
staining solution. Any of the components of the staining solution
can be added together or separately and in no particular order
wherein the resulting staining solution is added to the gel.
Alternatively, the components of the staining solution can be added
to a gel in a step-wise fashion. The fluorescent compound is
prepared by dissolving in a solvent, such as water, DMSO, DMF or
methanol, usually at a final concentration of about 0.1 .mu.M to
100 .mu.M, preferably the fluorescent compound is present in the
staining solution at a concentration of about 0.5 .mu.M to 20
.mu.M.
[0137] Analysis of the selectivity and specificity of the
fluorescent compounds for the poly-histidine affinity tags in a
SDS-polyacrylamide gel was evaluated as a function of pH.
Therefore, a preferred staining solution comprises an acid to
provide a moderatly acidic environment for the staining reaction.
An acidic environment is defined as a solution having a pH less
than 6.9. Typical suitable acidic components include without
limitation acetic acid, trichloroacetic acid, trifluoroacetic acid,
perchloric acid, phosphoric acid, or sulfuric acid. The acidic
component is typically present at a concentration of 1%-20%. The pH
of the staining mixture is preferably about pH 5-6.9 and most
preferred is about pH 6.5. The optimal pH for each compound used
may vary slightly depending on the compound used; for compound 1, 2
and 3 pH 6.5 is preferred. Alternatively, a neutral pH is also
desirable.
[0138] The pH of the staining mixture is optionally modified by the
inclusion of a buffering agent in addition to or in place of an
acidic component. In particular, the presence of a buffering agent
has been shown to improve staining of electrophoresis gels,
provided that an alcohol is included in the formulations as well.
Any buffering agent that maintains a mild acidic environment and is
compatible with the affinity tag and fusion protein in the sample
is suitable for inclusion in the staining mixture.
[0139] Useful buffering agents include salts of formate, acetate,
2-(N-morphilino) ethanesulfonic acid, imidazole,
N-2-hydroxyethyl-piperaz- ine-N'-2-ethanesulfonic acid (PIPES),
Tris (hydroxymethyl)aminomethane acetate, or Tris
(hydroxymethyl)aminomethane hydrochloride, 3-(N-morpholino)
propanesulfonic acid (MOPS). The family of Good's buffers,
including TRIS, MES, PIPES, MOPS, are preferred for the present
methods. An exemplified buffering agent is PIPES. The buffering
agent is typically present in the staining mixture at a
concentration of about 10 mM to 500 mM; preferably the
concentration is about 25 mM to 100 mM. These buffers are
particularly preferred for the non-covalent binding of a acetic
acid binding domain to the poly-histidine affinity tag because they
have pKa values that are similar to the pKa value of the imidazole
ring of the histidine residue.
[0140] Optionally, the staining solution may include a polar
organic solvent, typically an alcohol, to improve specific staining
of the affinity tag. The polar organic solvent, when present, is
typically included in the staining solution at a concentration of
5-50%. The presence of a polar organic solvent is particularly
advantageous when staining SDS-coated proteins, as is typically the
case when staining affinity tags that have been electrophoretically
separated on a SDS-polyacrylamide gel. Typically, SDS is removed
from a gel prior to staining by fixing, as described below, and
washing, however some SDS may remain and interfere with the
staining methods of the present invention. Without wishing to be
bound by any theory, it appears that the presence of an alcohol
improves the affinity of the fluorescent compound for the affinity
tag of a fusion protein by removing any SDS that was not removed by
the washing or fixing.
[0141] Optionally, the staining solution contains a metal ion salt.
This is particularly useful for staining solutions used to detect
poly-histidine affinity tags and calmodulin affinity tags. Nickel
ions and cobalt ions have affinity for both the acetic acid binding
domain of the present invention and the poly-histidine affinity
tag, therefore nickel or cobalt salts are optionally included in
staining solutions of the present invention. While the metal ions
do not improve the selective affinity or sensitivity of the binding
domain for the poly-histidine affinity tag the inclusion of the
metal ions is preferable for certain applications. For this reason,
a staining solution to be used to detect poly-histidine affinity
tags optionally includes nickel or cobalt ions. An exemplified salt
is nickel chloride but any nickel or cobalt salt known to one
skilled in the art can be used. The salt is typically present in
the staining solution at a concentration of about 10 nm to 1 mM;
preferably the concentration is about 1 .mu.M to 200 .mu.M.
[0142] Alternatively, some of the compounds of the present
invention, especially compounds 7-11, can be used to
calorimetrically detect poly-histidine affinity tag containing
fusion proteins when the staining solution comprises nickel ions at
a concentration about 10 .mu.M. Therefore, a preferred staining
solution for calorimetric applications comprises nickel ions at a
final concentration of about 10 .mu.M and typically any one of
compounds 7-11, See Example 19.
[0143] Calcium ions have an affinity for calmodulin, which
subsequently alters the conformation of the protein such that it
possesses affinity for the calmodulin affinity tag. Therefore, a
staining solution specific for calmodulin contains a calcium salt
along with a fluorescent compound that contains a fluorophore
covalently attached to the calmodulin protein.
[0144] 3. Fixing Solution
[0145] The fixing solution is required for optimal staining of
poly-histidine affinity tags that have been separated and
immobilized in an SDS-polyacrylamide gel. When fusion proteins are
denatured and separated on a polyacrylamide gel they become coated
with SDS, which masks the affinity tag such that the fluorescent
compound will not specifically or selectively bind to the affinity
tag. Therefore, the SDS must be removed prior to addition of the
staining solution.
[0146] The fixing solution contains a polar organic solvent,
typically an alcohol. Preferably, the polar organic solvent is an
alcohol having 1-6 carbon atoms, or a diol or triol having 2-6
carbon atoms. Preferred alcohols are methanol or ethanol mixed with
acetic acid. The alcohols are present in an aqueous solution of
about 50% ethanol or methanol with 10% acetic acid. Fixing
solutions containing less than 50% of ethanol or methanol generally
result in incomplete removal of SDS from the gels.
[0147] To remove the SDS coat from the immobilized fusion proteins,
the polyacrylamide gel is incubated in the fixing solution.
Preferably the gel is fixed in multiple sequential steps, typically
two. Essentially, the gel is immersed in the fixing solution for at
least 20 minutes and then removed from the solution and new
solution added for at least 3 hours and up to 24 hours. Generally,
one step of incubating the gel in fixing solution is insufficient
to remove all the SDS from the gel.
[0148] 4. Illumination
[0149] At any time after staining and during the washing step, the
sample is illuminated with a wavelength of light selected to give a
detectable optical response, and observed with a means for
detecting the optical response. Equipment that is useful for
illuminating the fluorescent compounds of the present invention
includes, but is not limited to, hand-held ultraviolet lamps,
mercury arc lamps, xenon lamps, lasers and laser diodes. These
illumination sources are optically integrated into laser scanners,
fuorescences microplate readers or standard or microfluorometers.
The degree and/or location of staining, compared with a standard or
expected response, indicates whether and to what degree the sample
possesses a given characteristic, i.e. fusion protein containing an
affinity tag.
[0150] The optical response is optionally detected by visual
inspection, or by use of any of the following devices: CCD camera,
video camera, photographic film, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, examination of the sample optionally includes sorting
portions of the sample according to their fluorescence
response.
III. Kits of the Invention
[0151] Suitable kits for detecting and selectively and
non-covalently labeling an affinity tag of a fusion protein also
form part of the invention. Such kits can be prepared from readily
available materials and reagents and can come in a variety of
embodiments. The contents of the kit will depend on the design of
the assay protocol or reagent for detection or measurement. All
kits will contain instructions, appropriate reagents and label, and
solid supports, as needed. Typically, instructions include a
tangible expression describing the reagent concentration or at
least one assay method parameter such as the relative amounts of
reagent and sample to be added together, maintenance time periods
for reagent/sample admixtures, temperature, buffer conditions and
the like to allow the user to carry out any one of the methods or
preparations described above.
[0152] Typically, kits useful for detecting an affinity tag of a
fusion protein that has been separated on a SDS-polyacrylamide gel
will include a staining solution. The kits will optionally include
affinity tag containing molecular weight markers, a fixing solution
and an additional detection reagent.
[0153] Typically, the affinity tag containing molecular weight
markers will be stained by the fluorescent compounds of the present
invention and are thus useful for estimating the size of the
detected fusion protein. This enables the end user to quickly
determine if a full-length fusion protein has been produced based
on the estimated molecular weight. A fixing solution, as described
above, is useful for removing the SDS from the polyacrylamide gel
as some of the compounds of the present invention will have minimal
affinity for the affinity tag in the presence of SDS. This is
particularly true for the fluorescent compounds that are used for
selectively binding to the poly-histidine affinity tag.
Alternatively, the end user may supply the fixing solution, as this
is made with reagents (alcohol) well known to one skilled in the
art.
[0154] Typically, an additional detection reagent will include a
total protein stain such as SYPRO.RTM. Ruby Dye and any
corresponding total protein stain disclosed in U.S. Pat. No.
6,316,276. Because SDS is removed by the fixing solution prior to
addition of the staining solution of the present invention, total
protein stains such as SYPRO Ruby are preferred because SDS is not
critical for the staining function. However, protocol changes can
be made when using a total protein stain that requires SDS for
staining sensitivity, such as SYPRO Orange Dye and SYPRO Red Dye,
by adding SDS back to the gel prior to a total protein stain step
and including SDS in the staining solution (Malone et al.
Electrophoresis (2001) 22(5):919-32). A preferred solution for
returning SDS back to a gel is 2% acid/0.0005% SDS, and optionally
40% ethanol, wherein the gel is incubated for at least one hour.
Alternatively, the total protein stain could be preformed prior to
detection of the affinity tag with the staining solution of the
present invention; therefore the SDS would not need to be added
back to the gel but simply removed prior to affinity tag detection
as contemplated by the present invention. Therefore, alternative
preferable total protein stains for gels are SYPRO Orange, SYPRO
Tangerine, SYPRO Red, Coomassie Fluor dyes or any corresponding dye
disclosed in U.S. Pat. Nos. 5,616,502 and 6,579,718. Alternative
total protein stains for gels include Coomassie Blue or silver
staining, staining techniques well known to those skilled in the
art.
[0155] The staining solution of the kit will depend on (1) the
affinity tag to be detected and (2) the desired absorption and
emission spectra from the fluorescent compound. The choice of the
binding domain dictates the particular affinity tag that will be
detected. As described above, particular binding domains of the
present invention have affinity for poly-histidine affinity tag,
poly-arginine affinity tag, GST affinity tag and calmodulin
affinity tag. The absorption and emission spectra of the
fluorescent compound is dictated by the fluorophore. The
fluorophores of the present invention cover almost the entire
spectrum of UV light, including the popular wavelengths 488, 532
and 633. Particularly useful fluorophores in fluorescent compounds
for detecting poly-histidine affinity tags are coumarin,
benzofuran, borapolyazaindacene, cyanine and xanthenes. Another
important aspect of the staining solution is the pH and the pKa
value wherein the optimal pH is dependent on the fluorescent
compound in the staining solution and the pKa value is dependent on
the affinity tag. Typically, a staining solution for detecting
poly-histidine affinity tags is mildly acidic or neutral, pH 5 to
7, and has a pKa of about 6.0 to about 7.5. Preferred is a pH about
6.5 and a pKa of about 6.8.
[0156] It is understood by one skilled in the art, that any of the
fluorescent compounds contemplated by the present invention can be
used to in a staining solution to be included in a kit. The
compounds are not intended to be limited to only the described
preferred embodiments.
IV. Applications
[0157] The compounds and methods described above for the
site-specific labeling of affinity tags has many applications and
is not simply limited to detection of affinity tags on a solid or
semi-solid matrix. One skilled in the art will appreciate many
other applications the fluorescent compound of the present
invention can be used in. For example, the fluorescent compounds
may be used to label affinity tag containing fusion protein in a
solution. This would serve the purpose for a quick determination
for the presence of the desired fusion protein or for more involved
applications wherein the fluorescent compound functions as a tracer
of the fusion protein in an in vitro assay. Such assays may
involve, but are not limited to, the study of protein-protein
interaction, signal transduction, post-translational modifications,
monitoring, metabolism and cell trafficking.
[0158] One skilled in the art will also recognize that live cell
(cell permeant) versions of the fluorescent compounds could be used
in a wide range of in vivo assays. Affinity tag containing fusion
proteins could be produced in an appropriate host cell, eukaryotic
or prokaryotic, and the fluorescent compounds of the present
invention could site-specifically label the intracellular fusion
proteins providing for a rigorous analysis of a protein of
interest. One could envision that this would be applicable for
determining drug targets or studying the functional proteome.
[0159] In one embodiment, modification of carboxylic groups with
acetoxymethyl (AM) ester groups results in uncharged molecules than
can penetrate cell membranes. Once inside the cells, the lipophilic
blocking groups are cleaved by nonspecific esterases revealing a
binding domain of the present invention, e.g., acetic acid binding
domain.
[0160] By way of example, the following present compound (Compound
13) has been derivatized to comprise three AM ester groups. 13
[0161] When the compound enters a cell the AM ester groups will be
cleaved revealing an acetic acid binding domain according to the
following structure (Compound 14). 14
[0162] Thus, the present compounds that comprise acetic acid
binding domains can be represent by the formula
--N(CH.sub.2COOR.sup.30) wherein R.sup.30 is the same or different
and is selected from the group consisting of hydrogen, salt ions,
an electron pair and CH.sub.2OCOCH.sub.3 (AM ester). In this way
the compounds of the present invention represent both cell permeant
and cell impermeant versions wherein for the live cell versions the
AM ester is cleaved unmasking the acetic acid binding domain.
[0163] Fluorogenic versions of the fluorescent compounds, i.e.,
version that demonstrate a detectable change upon non-covalently
binding to an affinity tag or compounds that are essentially
non-fluorescent until bound to an affinity tag, could be used in
certain applications. For example, the fluorogenic compounds could
be attached to a solid or semi-solid matrix and when an aliquot of
a sample thought to contain an affinity tag was added a change in
the detectable response would indicate the presence of an affinity
tag. Such solid or semi-solid matrix include without limitation,
multiwell plastic microplates, glass slides and arrays.
[0164] Additionally, some of the fluorescent compounds are also
calorimetric, especially compounds 7-10. These compounds can be
used in the same applications as the non-colorimetric compounds
however these compounds are especially useful for detecting
affinity tags in SDS-polyacrylamide gels and membrane blots. The
use of the calorimetric fluorescent compounds can be equally as
sensitive as detection by fluorescent wavelength and do not require
any special equipment for visualizing. The gels incubated with the
compounds can be inspected as one would with a Coomassie brilliant
blue stained gel to determine the presence of an affinity tag
containing fusion protein. (See, Example 19) A detailed description
of the invention having been provided above, the following examples
are given for the purpose of illustrating the invention and shall
not be construed as being a limitation on the scope of the
invention or claims.
EXAMPLES
Example 1
Synthesis of compound 1 [7-amino-3-(1-carboxy-1-(bis(carboxymethyl)
amino)-5-(acetylamino))pentyl-4-methylcoumarin-6-sulfonic acid,
tetratriethylammonium salt]
[0165] To a solution of
7-amino-3-((((succinimidyl)oxy)carbonyl)methyl)-4--
methylcoumarin-6-sulfonic acid (48 mg, 0.11 mmol) in DMF (3 mL) is
added a solution of NTA (34 mg, 0.13 mmol) and triethylamine (0.1
mL) in water (1 mL). The mixture is stirred at room temperature for
15 minutes and then concentrated to dryness in vacuo. The crude
product is purified on SEPHADEX LH-20, eluting with water to give
pure Compound 1 (59.3 mg). 15
Example 2
Synthesis of Compound 2
[4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-bi-
s((6-(propionyl)amino-2-bis(carboxymethyl)amino)hexanoic acid),
hexatriethylammonium salt]
[0166] To a solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dip- ropionic acid
(86 mg, 0.26 mmol) in DMF (2 mL) at 10.degree. C. is added
O-succinimidyl-N,N,N',N'-tetramethyluronium tetrafluoroborate (170
mg, 0,56 mmol) and triethylamine (0.087 mL). The mixture is stirred
at 10.degree. C. for 15 minutes and then followed by the addition
of a solution of NTA (160 mg, 0.61 mmol) and triethylamine (0.4 mL)
in water (2 mL). The mixture is stirred at 10.degree. C. for
another 30 minutes and then concentrated to dryness in vacuo. The
residue is purified on SEPHADEX LH-20 to give compound 2 (50 mg).
16
Example 2A
Synthesis of Compound 3
[0167] Compound 3 is synthesized similar to Compound 2 but with the
starting material
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-i-
ndacene-2,6-dipropionic acid 17
Example 3
Synthesis of Compound 4
[7-Hydroxy-6,8-difluoro-3-(1-carboxy-1-(bis(carbox-
ymethyl)amino)-5-(acetylamino))pentyl-4-methylcoumarin,
triethylammonium salt]
[0168] To a solution of
7-hydroxy-6,8-difluoro-4-methylcoumarin-3-acetic acid, succinimidyl
ester (44 mg, 0.12 mmol) in DMF (3 mL) is added a solution of NTA
(34.5 mg, 0.13 mmol) and triethylamine (0.1 mL) in water (1 mL).
The solution is stirred at room temperature for 30 minutes and then
concentrated to dryness in vacuo. The residue is purified on
SEPHADEX LH-20 to give compound 4 (40.9 mg). 18
Example 4
Synthesis of Compound 5
[7-Hydroxy-3-(1-carboxy-1-(bis(carboxymethyl)amino-
)-5-(acetylamino))pentyl-4-methylcoumarin]
[0169] To a solution of 7-hydroxy-4-methylcoumarin-3-acetic acid,
succinimidyl ester (141 mg, 0.427 mmol) in THF (5 mL) is added a
solution of NTA (74 mg, 0.282 mmol) and sodium bicarbonate (135 mg,
1.6 mmol) in water (5 mL). The mixture is stirred at room
temperature for 15 minutes and then acidified to pH=4 with 0.1 M
HCl. The solution is concentrated to dryness in vacuo and the
residue is purified on SEPHADEX LH-20, eluting with MeOH:water
(1:1) to give compound 5 (55 mg). 19
Example 5
Synthesis of compound 6
[7-dimethylamino-4-(1-carboxy-1-(bis(carboxymethyl-
)amino)-5-(acetylamino))pentylcoumarin, trisodium salt]
[0170] To a solution of 7-dimethylaminocoumarin-4-acetic acid,
succinimidyl ester (100 mg, 0.29 mmol) (1.5 mL) is added a solution
of NTA (38 mg, 0.145 mmol) and sodium bicarbonate (61 mg, 0.725
mmol) in water (1.5 mL). The mixture is stirred at room temperature
for 15 minutes and then concentrated to dryness in vacuo. The
residue is purified on SEPHADEX LH-20, eluting with methanol:water
(1:1) to give compound 6. 20
Example 6
Synthesis of Compound 7
[0171] To a solution of
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-ind-
acene-3-propionyl ethylenediamine, hydrochloride (BODIPYO FL EDA,
Molecular Probes 2390, 20 mg, 0.054 mmol) in 3 mL dry DMF under
argon is added DIEA (9 .mu.L, 0.054 mmol), followed by solid DTPA
anhydride (Aldrich, 77 mg, 0.22 mmol). The resulting orange mixture
is stirred at room temperature for 2 hours and then diluted with 5
mL water. The pH is raised to 9.0 with aqueous KOH. After another 2
hours, the reaction solution is concentrated in vacuo and the
product purified by column chromatography on Sephadex LH-20 using
E-pure water as eluant to give compound 7 as 22 mg of orange
powder. 21
Example 7
Synthesis of Compound 8
[0172]
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl
ethylenediamine, hydrochloride (BODIPY.RTM. FL EDA, Molecular
Probes 2390, 7 mg, 0.019 mmol) is dissolved into a mixture of
(S)-1-pisothiocyanatobenzyldiethylenetriaminepentaacetic acid (DTPA
isothiocyanate, Molecular Probes 24221, 10 mg, 0.019 mmol) in 2 mL
water. The pH (-3) is raised to 10 with aqueous sodium carbonate.
The resulting orange solution is stirred at room temperature for
3.5 hours, then concentrated in vacuo. The residue is purified by
column chromatography on Sephadex LH-20 using E-pure water as
eluant to give compound 8 as 29 mg of orange powder. 22
Example 8
Synthesis of Compound 9
[0173] For the synthesis of carbamate 9a a solution of
penta-t-butyl 1-(S)-(p-aminobenzyl)-diethylenetriamine-pentaacetate
(prepared according to the published procedure of Donald T. Corson
& Claude F. Meares. Bioconjugate Chem., 11 (2), 2000, 292-299,
0.800 g, 1.03 mmol) in 20 mL of methylene chloride is added 1 mL of
pyridine followed by the addition of a solution of the acid
chloride of N-CBZ-6-aminohexanoic acid (0.290 g, 1.02 mmol) in 5 mL
of methylene chloride. The reaction mixture is stirred overnight at
room temperature and concentrated in vacuo. The residue is
dissolved in 100 mL of ethyl acetate and the resulting solution is
washed with 10% HCl (2.times.30 mL), water (30 mL), brine (30 mL)
and dried over sodium sulfate. The solution is concentrated and put
on a silica gel column (packed with ethyl acetate). The column is
eluted first with ethyl acetate to remove impurities and then the
desired product is eluted with 10:1 chloroform-methanol. Pure
fractions are combined and the solvent evaporated to give amide 9a
(0.54 g, 54%) as a viscous oil.
[0174] For the synthesis of aminoacid 9b, the carbamate 9a (0.700
g, 0.683 mmol) is dissolved in 10 mL of TFA. The reaction mixture
is kept for 3 days at room temperature. Volatiles are evaporated in
vacuo and the residue is re-evaporated twice from toluene, leaving
a viscous oil. The oil is stirred with ethyl acetate until it
solidifies. The resulting solid is filtered and dried in vacuum to
give the aminoacid 9b (0.400 g, 96%).
[0175] For the synthesis of compound 9, the aminoacid 9b (0.090 g,
0.147 mmol) is suspended in 10 mL of water. The pH is adjusted to
pH .about.8 using 1 M KOH. The resulting solution is added to a
solution of
6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-in-
dacene-2-propionyl)amino)hexanoic acid, succinimidyl ester
(BODIPY.RTM. TMR-X, SE, MPI 6117, 0.03 g, 0.049 mmol) in 5 mL of
DMF. The reaction mixture is stirred overnight at room temperature.
The pH is monitored and adjusted to pH.about.8 during the first 2
hrs. The volatiles are removed in vacuo. The residue is
re-dissolved in water and put onto a Sephadex LH-20 column. The
column is eluted with E-pure water. Pure fractions containing the
most polar fluorescent product are combined. The resulting solution
is concentrated to .about.3 mL in vacuo and then lyophilized to
give Compound 9 as a red powder (0.061 g). 23
Example 9
Synthesis of Compound 10
[0176]
5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3--
yl)phenoxy)acetyl)amino) pentylamine, hydrochloride (BODIPY.RTM. TR
cadaverine, Molecular Probes 6251, 10 mg, 0.019 mmol) is dissolved
into a mixture of (S)-1-p
isothiocyanatobenzyldiethylenetriaminepentaacetic acid (DTPA
isothiocyanate, Molecular Probes 24221, 10 mg, 0.019 mmol) in 2 mL
water. The pH (-2) is raised to 10 with aqueous sodium carbonate.
The resulting blue solution is stirred at room temperature for two
days, then concentrated in vacuo. The residue is purified by column
chromatography on Sephadex LH-20 using E-pure water as eluant to
give compound 10 as 2 mg of purple powder. 24
Example 10
Synthesis of BODIPY FL-TTHA Compound 11
[0177] To a solution of
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-ind-
acene-3-propionyl ethylenediamine, hydrochloride (BODIPY.RTM. FL
EDA, Molecular Probes 2390, 20 mg, 0.054 mmol) in 3 mL dry DMF
under argon is added DIEA (9 .mu.L, 0.054 mmol), followed by solid
TTHA anhydride (prepared according to Achour et al., Inorganic
Chemistry 1998, 37: 2729-2740,100 mg, 0.22 mmol). The resulting
orange mixture is stirred at room temperature for 2 hours, then
diluted with 5 mL water. The pH is raised to 9.0 with aqueous KOH.
After another 2 hours, the reaction solution is concentrated in
vacuo and the product purified by column chromatography on Sephadex
LH-20 using E-pure water as eluant to give compound 11 as an orange
powder. 25
Example 11
Synthesis of Compound 13
[0178] N.alpha.,N.alpha.-Bis(carboxymethyl)lysine (0.157 g, 0.600
mmol) was dissolved in a mixture of 4.8 mL 1M
Et.sub.3NH.sub.2CO.sub.3 buffer and 15 mL water.
4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacen-
e-3-propionic acid, succinimidyl ester (BODIPY.RTM. 576/589 SE,
0.170 g, 0.401 mmol) was dissolved in 30 mL of dioxane and added to
the amino acid solution. The reaction mixture was stirred for 1 h
at RT and evaporated to dryness. The residue was re-evaporated from
water to remove tetraethylammonium salts. The crude product was
dissolved in water and loaded onto an LH-20 column (packed in
water). The column was eluted with water. Fractions containing pure
material were combined and lyophilized to give compound 13 as a
dark red powder (0.120 g, 34%) as its triethylammonium salt.
Example 12
Synthesis of Compound 14
[0179] The triethylammonium salt 13 (0.120 g, 0.137 mmol) was
suspended in 5 mL of DMF. i-Pr.sub.2NEt (0.14 mL, 0.82 mmol) was
added to the suspension followed by BrCH.sub.2OAc (0.08 mL, 0.8
mmol). The reaction mixture was stirred for 4 hrs at RT and then
diluted with brine (30 mL). The product was extracted with ethyl
acetate (3.times.30 mL). The combined extracts were washed with
water (3.times.30 mL), brine (30 mL), dried over anhydrous sodium
sulfate and evaporated. The crude product was dissolved in
chloroform and loaded onto a silica gel column packed with 4:8:0.1
chloroform-ethyl acetate-acetic acid. The same solvent mixture was
used to elute the column. Fractions containing pure product were
combined and evaporated in vacuo. The residue was re-evaporated
from toluene to give AM ester 14 as a dark purple wax (0.081 g,
75%).
Example 13
Synthesis of Compound 15
[0180] p-Nitrophenylalanine methyl ester hydrochloride 15a (Bachem,
cat. # F-1910; 2.00 g, 7.68 mmol) was added portionwise to 9.9 mL
(92 mmol) of diethylenetriamine with stirring at RT. When all
hydrochloride was added the mixture was stirred for additional 5
hrs at RT. Excess of diethylenetriamine was removed in vacuum. The
residue was dissolved in 20 mL of conc. ammonia solution and the
product was extracted with CH.sub.2Cl.sub.2 (10.times.20 mL). The
combined extracts were dried over sodium sulfate and concentrated
in vacuum to give amide 15b (1.98 g, 87%) as yellow oil.
[0181] Amide 15b (1.98 g, 6.71 mmol) was dissolved in 60 mL of dry
THF. BH.sub.3.THF complex (1M solution of in THF, 60.4 mL, 60.4
mmol) was added to amide 15b dropwise under nitrogen with stirring
and cooling (ice/water bath). After all amount of complex was
added, the temperature was allowed to rise to ambient and the
mixture was stirred under reflux for 15 hrs. Then the mixture was
cooled again (ice/water bath) and excess of BH.sub.3 was carefully
decomposed with water (5 mL, dropwise, stirring). The resulting
solution was concentrated in vacuum and the residue mixed with 35
mL of water and 35 mL of conc. HCl. The solution was stirred for
3.5 hrs under reflux then 20 hrs at RT and evaporated to dryness.
The residue was mixed with 50 mL of conc. ammonia and 50 mL of
water. The product was extracted with chloroform (6.times.100 mL).
The combined extracts were dried over Na.sub.2SO.sub.4 and
evaporated to give amine 15c (1.37 g, 73%) as yellow oil.
[0182] Amine 15c (1.37 g, 4.88 mmol) was dissolved in 50 mL of DMF.
Diisopropyethylamine (12.7 mL, 72.9 mmol) and tert-butyl
bromoacetate (8.64 mL, 58.5 mmol) were added to the solution,
followed by addition of powdered KI (0.89 g, 5.4 mmol). The
reaction mixture was stirred for 72 hrs at RT and evaporated to
dryness. The residue was mixed with 100 mL of water and the product
extracted with diethyl ether (3.times.40 mL). The combined extracts
were washed with water (40 mL), brine (40 mL), dried over sodium
sulfate and evaporated. The crude product was dissolved in 2:1
hexanes--ethyl acetate mixture and loaded on silica gel column
(packed with 2:1 hexanes--ethyl acetate mixture). The same solvent
mixture was used to elute the column. Pure fractions were combined
and evaporated to give hexaester 15d as yellow oil (1.68 g,
36%).
[0183] Ester 15d (1.68 g, 1.74 mmol) was dissolved in 50 mL of
methylene chloride. 10% Pd/C (100 mg) was added to the solution and
the mixture was shaken in Parr Apparatus at 50 psi for 4 hrs. The
catalyst was filtered off, and the solution vas concentrated in
vacuum. The residue was dissolved in 9:1 CH.sub.3CN:water mixture
and the solution was loaded on silica gel column (packed with 9:1
CH.sub.3CN:water mixture). The column was eluted with the same
solvent mixture. Pure fractions were combined and concentrated in
vacuum to give amine 15e as yellow oil (1.54 g, 95%).
[0184] N-CBZ-6-aminohexanoic acid 15f (0.600 g, 2.26 mmol) was
dissolved in 5 mL of methylene chloride. DCC (0.234 g, 1.13 mmol)
was added to the solution and reaction mixture was stirred over
weekend at RT. The precipitate was filtered off and washed with 2
mL of methylene chloride. Methylene chloride solutions were
combined and evaporated to give anhydride 15g (0.58 g, quant.).
[0185] Amine 15e (0.600 g, 0.640 mmol) was dissolved in 5 mL of
DMF. i-Pr.sub.2NEt (0.54 mL, 3.1 mmol) was added to the solution
followed by addition of anhydride 15g (0.58 g, 1.13 mmol) as a
solution in 3 mL of DMF. The reaction mixture was stirred overnight
at RT. The solution was diluted with 80 mL of 0.5M KOH and the
product was extracted with EtOAc (3.times.40 mL). The combined
extracts were washed with water (3.times.30 mL), brine (30 mL),
dried over sodium sulfate and evaporated. The crude product was
suspended in 1:1 hexanes--EtOAc mixture and loaded on silica gel
column (packed with 2:1 EtOAc--hexanes mixture. The column was
eluted first with 2:1 EtOAc--hexanes mixture and then with 10% MeOH
in chloroform. Fractions containing pure amide were combined and
evaporated to give desired amide 15 h as viscous oil (0.846 g).
[0186] CBZ protected amide 15h (0.84 g, 0.71 mmol) was dissolved in
5 mL of TFA. The solution was kept at RT for 72 hrs, and then
evaporated in vacuum. The residue was re-evaporated from toluene
(3.times.20 ML) and triturated with EtOAc. White precipitate
formed. Mixture was centrifuged, supernatant separated and solid
washed with fresh EtOAc. Mixture was stirred and centrifuged again.
After supernatant was removed the procedure was repeated three more
times and then the product was dried in vacuum to give amine 15i as
a white solid (0.521 g, 89%).
[0187] Amine 151(0.054 g, 0.065 mmol) was dissolved in 4 mL of DMF.
i-Pr.sub.2NEt (0.023 mL, 0.13 mmol) was added to the solution.
White precipitate formed. Water was added to the solution (about 2
mL) until all solid dissolved. SE ester D6117 (0.02 g, 0.033 mmol)
was dissolved in 2 mL of DMF and two solutions were mixed. After
stirring for 20 min. 0.5 g of sodium bicarbonate was added to the
solution and the reaction mixture was stirred for 48 hrs at RT.
[0188] Reaction mixture was concentrated in vacuum, the residue
dissolved in water (4 mL) and loaded on LH-20 column. The column
was eluted with water. Fractions containing most polar fluorescent
product were combined and concentrated to the volume .about.10 mL.
Solution was acidified with 1 mL of 10% HCl and the product was
extracted with n-BuOH (3.times.10 mL). The combined extracts were
concentrated in vacuum and the residue was mixed with 10 mL of
water. The solid was filtered off, washed with water (2 mL) and
dissolved in 5% ammonia (2 mL). The resulting solution was loaded
on LH-20 column ant eluted with water. Pure fractions were combined
concentrated in vacuum and lyophilized to give amide 15 (0.014 g of
Compound 15, isomer a and 0.022 g of Compound 15, isomer b. Both
fraction show similar purity by LCMS). MS+H: 1257 (calculated for
C.sub.58H.sub.78N.sub.9O.sub.16BF.sub.2.3NH.sub.3: 1256). 26
Example 14
Synthesis of Compound 16
[0189] NTA (0.100 g, 0.382 mmol) was dissolved in 2 mL of water.
The pH of the solution was adjusted to 8 using 1M KOH. (D6117,
Molecular Probes, Inc.) (0.100 g, 0.164 mmol) was dissolved in 2 mL
of DMF and added to solution of amino acid. The reaction mixture
was stirred for 2 hrs at RT. During the reaction pH was monitored
and adjusted to 8 with 1M KOH. After all SE ester (D6117) was
consumed the reaction mixture was evaporated. The residue was
dissolved in water and solution was loaded on LH-20 column. The
column was eluted with water. Fractions containing pure product
(TLC, A/B 1:1) were combined, concentrated to the volume of 2-3 mL,
and lyophilized to give Compound 16 (0.130 g, 91%). [MS-H] 754.3,
calculated for C.sub.37H.sub.48N.sub.5O.sub.9BF.sub.2 755.6.
Solution A: dioaxane:i-PrOH:water:ammonia 80:40:68:72. Solution B:
dioaxane:i-PrOH:water:ammonia 15:58:13:14 27
Example 15
Detection of Fusion Proteins Containing a Poly-Histidine Affinity
Tag in Polyacrylamide Gels
[0190] Eschericia coli BL21 DE3 cells were transformed with
plasmids containing either the human ATP synthase .alpha. subunit,
the d subunit (including the leader sequence) or urate oxidase.
Both proteins were constructed to have a poly-histidine affinity
tag comprising six histidine residues, at the N-terminus and could
be induced by isopropyl-beta-D-thiogalactoside (IPTG) addition to
the medium. Pre-cultures (10 ml) were grown overnight in bacterial
cell culture medium (LB medium) at 37.degree. C. with constant
shaking. The next day 100 .mu.l was transferred to 50 ml of fresh
LB medium containing 0.1 mg/ml ampicillin and grown until they
reached an optical density at 595 nm (OD.sub.595) of 0.8. At this
point 5 ml of culture was removed and immediately frozen on dry
ice. To the rest of the culture 0.8 mM IPTG was added to induce the
over-expression of the subunits. Samples (5 ml each) were taken
after 10 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, and 3 h and again
frozen on dry ice.
[0191] The cells from the different time points were pelleted (at
5000.times.g) and the supernatant was discarded. The cells were
lysed adding 200 Id of buffer 1 (0.3% SDS, 200 mM DTT, 28 mM Tris
base, 28 mM Tris HCl, pH 8.0) and incubated for 10 min, followed by
a short (2 min) sonication to break the cells open completely. To
remove the DNA, 20 .mu.l of buffer 11 (24 mM Tris Base, 476 mM Tris
HCl, 50 mM MgCl.sub.2, 1 mg/ml DNAse 1, 0.25 mg/ml RNAse A) was
added and the cell extract was incubated for another 10 min.
Finally, 100 .mu.l of the cell extract was removed and mixed with
40 .mu.l of 5.times.SDS sample buffer (290 mM Tris, 25% glycerol,
250 mM DTT, 10% SDS, 0.01% bromophenol blue). After vortex mixing,
the samples were centrifuged at maximum speed
(.about.12,000.times.g) in a microcentrifuge and the supernatant
was subjected to SDS-polyacrylamide gel electrophoresis.
[0192] Proteins were separated by SDS-polyacrylamide gel
electrophoresis utilizing 13% T, 2.6% C gels. % T is the total
monomer concentration expressed in grams per 100 ml and % C is the
percentage crosslinker. The 0.75 mm thick, 6.times.10 cm gels were
subjected to electrophoresis using the Bio-Rad mini-Protean III
system according to standard procedures.
[0193] Following separation of the proteins on a SDS-polyacrylamide
gel, the gels were fixed for 20 minutes in 100 ml of 50% ethanol/7%
acetic acid and then fixed overnight in 100 ml of fresh fixative
solution to ensure complete elimination of SDS. Gels were next
washed 3 times for 20 minutes each in deionized water. The gels
were then incubated in a staining solution containing 10 .mu.M
compound 1 or 2 .mu.M compound 2; 100 .mu.M NiCl.sub.2; 50 mM PIPES
at pH 6.5 for 45-90 minutes in a total volume of 25 ml. Afterwards,
the gels were washed 2 to 4 times for 20 minutes each in deionized
water. In order to ensure that the optimal signal was documented,
gels were imaged after the second and fourth wash.
[0194] The resulting blue-fluorescent signal produced by compound 1
was visualized using 300 nm trans-illumination and 520 nm band pass
emission filter on the Lumi-Imager (Roche Biochemicals,
Indianapolis, Ind.), a cooled CCD-camera based system digitizing at
1024.times.1024 pixels resolution with 16-bit gray scale levels
assigned per pixel. Alternatively, the signal was visualized
utilizing a UVP transilluminator/Polaroid MP4+ camera system (UVP,
Upland, Calif.) with 365 nm transillumination and photographed with
Polaroid 667 black-and-white print film using a SYPRO.RTM. protein
gel stain photographic filter (Molecular Probes, Eugene,
Oreg.).
[0195] The resulting green-fluorescent signal produced by compound
2 was visualized using the 473 nm excitation line of the SHG laser
on the Fuji FLA-3000G Fluorescence Image Analyzer (Fuji Photo,
Tokyo, Japan) with the 520 nm long pass filter or the 580 nm band
pass filter, respectively. See, FIGS. 1 and 2.
Example 16
Detection of Fusion Proteins Containing a Poly-Histidine Affinity
Tag in Polyacrylamide Gels that are First Separated by Isolelectric
Focusing
[0196] E. coli cultures of induced and un-induced human ATP
synthase d subunit (100 ml each) were grown as described in Example
11 and the cells were pelleted at 5000.times.g. The cells were
resuspended in 2 ml of 25 mM Tris, pH 7.5 before addition of 4 ml
of 28 mM Tris base, 22 mM Tris HCl, 0.3% SDS to lyse the cells.
After 5 minutes, a sufficient amount of 1M MgCl.sub.2 was added to
make a final concentration of 5 mM, followed by 10 .mu.l RNAse A
(10 mg/ml) and 40 .mu.l DNAse 1 (10 mg/ml) to digest the nucleic
acids. The raw cell extract was then mixed with 6 ml Urea buffer (7
M Urea, 2 M Thiourea, 2% Chaps, 1% Zwittergent 3-10, 65 mM DTT) and
insoluble material was pelleted by centrifigation (15,000.times.g,
SS34 rotor). The supernatant was then injected into the Rotofor
chamber (Bio-Rad Laboratories, Hercules, Calif.) according to the
manufacturers manual using the same urea buffer in the chamber. The
proteins were focused for roughly 3 h before harvesting into 20
fractions spanning a pH range of 2-12. Fractions were collected
using the system's vacuum manifold and were acetone-precipitated
and resuspended in SDS-sample buffer. For SDS polyacrylamide gel
electrophoresis 30 .mu.l of sample per fraction was utilized and
gels were subsequently stained for the presence of the
oligopoly-histidine affinity tag using Compound 2 as described in
Example 11.
Example 17
Serial Dichromatic Detection of Poly-Histidine Affinity Tag and
Total Protein in SDS-Polyacrylamide Gels
[0197] Following selective staining of the poly-histidine affinity
tag containing fusion proteins separated on a SDS-polyacrylamide
gel, as described in Example 15, the gel was incubated overnight
with SYPRO.RTM. Ruby protein gel stain with gentle orbital shaking,
typically 50 rpm. The gel was then incubated in 7% acetic acid, 10%
methanol for 30 minutes, also at 50 rpm. The fluorescent signal
from the affinity tag containing proteins and non-affinity tag
proteins was collected with a standard CCD camera-based imaging
system with 300 nm UV light excitation and a 600 nm bandpass
filter.
Example 18
Detection of Poly-Histidine Affinity Tag Containing Fusion Proteins
in Two-Dimensional Polyacrylamide Gels.
[0198] E. coli BL21 DE 3 cells expressing poly-histidine affinity
tag ATP synthase d subunit induced with IPTG were prepared and a
lystate (100 .mu.l) was diluted in urea buffer (2 M Thiourea, 7 M
Urea, 2% CHAPS, 1% Zwittergent 3-10, 0.8% Ampholytes 3-10, 56 mM
DTT) and applied on a first dimension IPG strip (3-10 non linear,
18 cm; Amersham Pharmacia) that had been rehydrated overnight in
urea buffer. The strips were overlayed with 2 ml of light mineral
oil and the proteins focused for 24.5 h, at 70 kVh and 20.degree.
C. for a final voltage of 100 nA/strip. The IPG strips were
equilibrated in 300 mM Tris/Base, 75 mM Tris/HCl, 3% SDS, 50 mM
DTT, 0.01% Bromophenol Blue for 10 min and then laid on top of a
12.5% SDS-polyacrylamide gel. Electrophoresis was performed
according to standard procedures for 4.5 h.
[0199] After the second dimension electrophoresis the gels were
fixed in 10% ethanol, 7% acetic acid overnight to remove SDS. The
next day the gels were washed twice with dH.sub.2O for 20 minutes
each before equilibration in 50 mM PIPES, 1 mM NiCl.sub.2, pH 6.5.
The gels were washed again twice for 15 minutes each before
staining with 10 .mu.M Compound 1 in 50 mM PIPES, pH 6.5 (250 ml).
To remove excess dye the gels were washed twice in dH2O for 20
minutes each. The staining was imaged on a Lumi-Imager (Roche)
using UV light excitation and a 520 nm emission filter with a 5 s
exposure time.
[0200] Following detection of poly-histidine affinity tag
containing fusion proteins, the gels was stained for total protein
using SYPRO.RTM. Ruby protein gels stain as described in Example
17.
Example 19
Detection of Fusion Proteins Containing a Poly-Histidine Affinity
Tag in Polyacrylamide Gels Using a Colorimetric Fluorescent
Compound
[0201] Fusion proteins containing a poly-histidine affinity tag
were prepared and separated from Eschericia coli lysate proteins by
SDS-polyacrylamide gel electrophoresis as described in Example 15.
Following separation of the proteins on a SDS-polyacrylamide gel,
the gels were fixed for 20 minutes in 100 ml of 50% ethanol/7%
acetic acid and then fixed overnight in 100 ml of fresh fixative
solution to ensure complete elimination of SDS. Gels were next
washed 3 times for 20 minutes each in deionized water. The gels
were then incubated in a staining solution containing 10 .mu.M
compound 9 or compound 10; 10 .mu.M NiCl.sub.2; 50 mM PIPES at pH
6.5 for 45-90 minutes in a total volume of 25 ml. Afterwards, the
gels-were washed 2 to 4 times for 20 minutes each in deionized
water. The colorimetric signal from the poly-histidine affinity tag
containing proteins was detected with a standard CCD camera-based
imaging system with white light illumination and no filter
according to standard Coomassie Blue or silver staining imaging
methods.
Example 20
Detection of Glutathione S-Transferase (GST) with Texas Reds
X-Glutathione Compound in Polyacrylamide Gels
[0202] A purified sample of GST was separated by SDS-polyacrylamide
gel electrophoresis utilizing 13% T, 2.6% C gels. % T is the total
monomer concentration expressed in grams per 100 ml and % C is the
percentage crosslinker. The 0.75 mm thick, 6.times.10 cm gels were
subjected to electrophoresis using the Bio-Rad mini-Protean III
system according to standard procedures.
[0203] Following separation of the protein on a SDS-polyacrylamide
gel, the gel was fixed for 1 hour in 100 ml of 50% methanol/10%
acetic acid and then fixed overnight in 100 ml of fresh fixative
solution to ensure complete elimination of SDS. Gels were next
washed 3 times for 20 minutes in deionized water. The gels were
then incubated in a staining solution containing 5 .mu.M Texas Red
X-glutathione compound in 50 mM PIPES at pH 6.5 for 90 minutes in a
total volume of 50 ml. Afterwards, the gels were washed 2 times for
20 minutes each in deionized water.
[0204] The resulting red-fluorescent signal produced by Texas
Red-glutathione was visualized using the 532 nm excitation line of
the SHG laser on the Fuji FLA-3000G Fluorescence Image Analyzer
(Fuji Photo, Tokyo, Japan) and 580 band pass emission filter. See,
FIG. 4.
Example 21
Detection of Fusion Proteins Containing a Poly-Histidine Affinity
Tag on a Membrane Blot
[0205] Escherichia coli lysates containing 6.times.histidine-tagged
A subunit of ATPase and 6.times.histidine-tagged porin are
fractionated by 13% T, 0.8% C SDS-polyacrylamide gel
electrophoresis and electroblotted onto PVDF membrane. Blots are
wetted with 100% methanol and then fixed with 50% methanol/7%
acetic acid, briefly rinsed in deionized water and then stained for
15 minutes with either Pro-Q Sapphire 488 or Pro-Q Sapphire 532 gel
stain solution. Blots are destained with two five-minute washes in
50 mM PIPES, pH 6.5, 20% acetonitrile to obtain fairly specific
detection of the two his-tagged proteins. Blots are briefly washed
in water and then dried before imaging. With both dyes, the two
oligohistidine-tagged proteins are readily distinguished from other
proteins in the lysate as brightly fluorescing bands. Limits of
detection are approximately 20 ng.
Example 22
Detection of Fusion Proteins Containing a Poly-Histidine Affinity
Tag on a Microarray
[0206] Purified oligohistidine-tagged fusion proteins (the a
subunit of Escherichia coli ATPase and porin), as well as control
proteins (bovine serum albumin and ovalbumin) are arrayed from a
source plate (384 well plate) concentration of 0.468 .mu.g/ml-0.240
mg/ml in water, onto HydroGel coated slides (Perkin Elmer), using
the BioChip Arrayer.TM. (Perkin Elmer). The BioChip Arrayer.TM.
utilizes a PiezoTip.TM. Dispenser consisting of 4 glass
capillaries. Proteins are dispensed from the PiezoTip.TM. by
droplets 333 pl in volume to create array spots -200 microns in
diameter with a 500 micron horizontal and vertical pitch
(pitch=center to center spacing of spots). Proteins are arrayed in
duplicate in four rows, with 10 dilution points. The resulting
concentration range of the array is 166.5 pg/spot-0.325 pg/spot.
For detection of oligohistine-tagged proteins, slides are incubated
for 45 minutes on a rotator in 50% ethanol/7% acetic acid and then
fixed overnight in fresh fixative solution to ensure complete
elimination of SDS. Microarrays are next washed 3 times for 20
minutes each in deionized water. The microarrays are then incubated
in a staining solution containing 10 .mu.M Compound 2 or Compound
15; 50 mM PIPES at pH 6.5 for 45-90 minutes. Afterwards, the
microarrays are washed 2 to 4 times for 20 minutes each in
deionized water. In order to ensure that the optimal signal was
documented, gels are imaged after the second and fourth wash.
Slides are then spun briefly in a microarray high-speed centrifuge
affixed with a rotor with a slide holder (Telechem) at -6000 rpm to
remove excess liquid. After slides are dry, the arrays are imaged
using the ScanArray.RTM. 5000 XL Microarray Analysis System
(Packard Instrument Co., Meriden, Conn.) using the 488 nm laser and
522 nm emission filter. The oligohistine tagged proteins are
detected as discrete fluorescent spots, while little or no signal
generated on the control proteins. Detection sensitivity is less
than 20 pg.
Example 23
Detection of Fusion Proteins Containing Poly-Arginine Affinity Tag
in a Polyacrylamide Gel
[0207] An Escherichia coli lysate containing an expressed
oligo-arginine-tagged fusion protein (porin) is separated by
SDS-polyacrylamide gel electrophoresis utilizing 13% T, 2.6% C
gels. % T is the total monomer concentration expressed in grams per
100 ml and % C is the percentage crosslinker. The 0.75 mm thick,
6.times.10 cm gels are subjected to electrophoresis using the
Bio-Rad mini-Protean III system according to standard procedures.
Following separation of the proteins on a SDS-polyacrylamide gel,
the gels are fixed for 20 minutes in 100 ml of 50% ethanol/7%
acetic acid and then fixed overnight in 100 ml of fresh fixative
solution to ensure complete elimination of SDS. Gels are next
washed 3 times for 20 minutes each in deionized water. The gels are
then incubated in a staining solution containing 10 .mu.M compound
1 or 2 .mu.M compound 2; 100 .mu.M NiCl.sub.2; 50 mM PIPES at pH
6.5 for 45-90 minutes in a total volume of 25 ml. Afterwards, the
gels are washed 2 to 4 times for 20 minutes each in deionized
water. In order to ensure that the optimal signal is documented,
gels are imaged after the second and fourth wash.
[0208] The preceding examples can be repeated with similar success
by substituting the specifically described fluorescent compound,
affinity tag and staining conditions of the preceding examples with
those generically and specifically described in the forgoing
description. One skilled in the art can easily ascertain the
essential characteristics of the present invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt to various
usages and conditions.
[0209] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *