U.S. patent application number 10/818694 was filed with the patent office on 2005-04-28 for use of self-assembled monolayers to probe the structure of a target molecule.
This patent application is currently assigned to Minerva Biotechnologies, Inc.. Invention is credited to Bamdad, Cynthia Carol.
Application Number | 20050089931 10/818694 |
Document ID | / |
Family ID | 32072686 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089931 |
Kind Code |
A1 |
Bamdad, Cynthia Carol |
April 28, 2005 |
Use of self-assembled monolayers to probe the structure of a target
molecule
Abstract
Weak binding motifs were transformed into a high affinity ligand
surface by using a heterologous self-assembled monolayer (SAM) as a
rigid scaffold to present discrete binding moieties, in a
controlled geometry, to a target molecule. At a critical ligand
density, the discrete binding moieties simulated a multivalent
ligand and promoted high-affinity, cooperative binding of the
target molecule. Statistical calculations were applied to SAM
components in solution and gold-sulfur packing dimensions to
extract the inter-ligand-distance within the SAM. This distance
information is valuable to the rational design of multivalent
drugs.
Inventors: |
Bamdad, Cynthia Carol;
(Newton, MA) |
Correspondence
Address: |
Timothy J. Oyer, Ph.D.
Wolf, Greenfield & Sacks, P.C.
600 Atlantic Avenue
Boston
MA
02210
US
|
Assignee: |
Minerva Biotechnologies,
Inc.
Newton
MA
|
Family ID: |
32072686 |
Appl. No.: |
10/818694 |
Filed: |
April 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10818694 |
Apr 6, 2004 |
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09324258 |
Jun 2, 1999 |
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6723517 |
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60087766 |
Jun 2, 1998 |
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Current U.S.
Class: |
435/7.1 ;
436/518 |
Current CPC
Class: |
B82Y 30/00 20130101;
G01N 33/553 20130101; G01N 33/5432 20130101; G01N 2610/00
20130101 |
Class at
Publication: |
435/007.1 ;
436/518 |
International
Class: |
G01N 033/53; G01N
033/543 |
Goverment Interests
[0002] This work was supported, in part, by NIH Grants
5T32EM-07598-18 and GM-32308. The government of the United States
of America may have some rights in this invention.
Claims
What is claimed is:
1. A method for presenting discrete binding moieties in a
controlled geometry that promotes high-affinity, cooperative
binding to a target molecule, comprising forming a self-assembled
monolayer that incorporates at least one thiol species (the
biospecific component) that is capable of directly or indirectly
displaying a binding partner to said target molecule and at least
one inert spacer thiol component by the process of: (a) mixing the
biospecific and a second component of said self-assembled monolayer
in defined proportions; and (b) forming said self-assembled
monolayer on a suitable substrate.
2-39. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/324,258, filed Jun. 2, 1999, which claims
priority to U.S. Provisional Patent Application No. 60/087,766,
filed on Jun. 2, 1998.
FIELD OF INVENTION
[0003] The present invention relates to the use of self-assembled
monolayers attached to surfaces for the detection and probing of
target molecule structure and function.
BACKGROUND OF THE INVENTION
[0004] Combinatorial chemistry techniques are used to synthesize
diverse "libraries" of unique chemical compounds. These small
molecule libraries often yield drug candidates that are capable of
binding a specific biological target but because of their small
size and relative simple chemical makeup, they characteristically
interact with the target in a low affinity interaction. These low
affinity interactions cannot adequately compete with larger more
diverse natural ligands, like proteins and protein complexes, and
thus provide little therapeutic value. Natural products, which are
naturally occurring organisms isolated from soils, yeast, marine
organisms, and the like are larger and chemically more interesting
than small molecules from combinatorial libraries. Natural products
are routinely screened for therapeutic activity against
disease-related organisms. Many cancer drugs have been identified
in this way. The problem with developing a natural product for the
drug marked is that they are large and chemically complicated,
which means that elaborate and expensive schemes for their
synthesis must be developed. Identifying a synthetic scheme that is
commercially feasible is a technical challenge that at best takes
years and millions of dollars to accomplish and at worst cannot be
done. For this reason, there is interest in enhancing the affinity
between small molecule drugs and their biologically relevant
targets.
[0005] Knowles and colleagues, at Harvard, reported that they could
enhance the binding affinity of a small molecule for a particular
target by attaching a "greasy tail" to the small molecule. This
hydrophobic tail was later shown to interact with a hydrophobic
patch on the target molecule adjacent to the binding site.
[0006] Many biologically relevant target molecules present more
than one binding site for a particular ligand. Some present pseudo
identical binding sites with which they bind natural ligands that
contain "repeats" of a binding motif. It is known that bivalent
interactions (like antibody interactions) are higher affinity
interactions than monovalent interactions, due to the cooperative
binding effect. Therefore, one would like to link several small
molecule drugs together to form a pseudo multivalent drug that
would interact more strongly with a multi-binding-site target
molecule. The problem with this logic is that the enthalpic
advantage of the additional binding energy is offset by the large
entropic energy cost of ordering the connected binding moieties.
However, making the linker between the binding moieties a rigid
linker would introduce order and thus minimize the entropic cost to
yield a higher affinity interaction. In order to connect two
binding moieties (the small molecule drugs) with a rigid linker, in
a geometry that would encourage its binding to the target molecule,
one would need to know apriori the distance between the binding
sites on the target molecule. This inter-binding-site distance
information is currently derived from X-ray or NMR structure
determination of the target molecule. This process is
time-consuming (years) and expensive.
[0007] The subject of this invention is how self-assembled
monolayers (SAMs) can be used to present discrete binding moieties,
at varying densities, in a rigid 2-dimensional array, to
multivalent target molecules in order to promote a higher affinity,
cooperative interaction. Ligand densities within the SAM are varied
to determine the critical distance between binding moieties that
will promote simultaneous, cooperative binding of the target
molecule. By monitoring the kinetics of binding events between the
target molecule and the variable density ligand surfaces, one can
empirically determine the lowest surface density that prompts a
large shift in affinity for the multivalent target molecule. One
can then use Poisson statistics to infer the distance between
surface-immobilized ligands and thus also the distance between the
binding sites on the target molecule. Once this distance
information has been deduced, it can be used to rationally design
bi- or multi-valent drugs or rigid linkers to connect two binding
moieties. Alternatively, the SAM itself can become a part of the
"drug"; in this case, the SAM is used as the "rigid linker" between
binding moieties to present multiple binding motifs, at the
empirically determined critical density, to promote the higher
affinity cooperative interaction. The SAM, presented ligands and
underlying gold (may be gold colloids) are both the drug and the
drug delivery system. Inert thiols of the SAMs can be terminated
with lipid-like groups to facilitate drug delivery. Similarly, a
biospecific ligand could be incorporated (at varying densities)
into a liposome, at the critical presentation density determined,
and used directly as a multivalent drug in its own delivery
system.
SUMMARY OF THE INVENTION
[0008] Self-assembled monolayers are used as a rigid 2-dimensional
matrix for presenting binding moieties, at varying distances from
each other, to a target molecule. Two-component SAMs incorporate an
inert spacer molecule and a biospecific molecule that can directly
or indirectly present a binding moiety to a target molecule. The
distance between the biospecific molecules in the array, the ligand
density, is controlled by manipulating the concentrations of the
two component thiols in solution before deposition onto gold. The
affinity of the interaction between the surface immobilized ligands
and the multivalent target molecule is monitored as a function of
ligand density. The lowest ligand surface density that elicits a
jump in affinity for the target molecule contains the critical
information needed to extract the distance between binding sites on
the target molecule. The dimensions of the hexagonal tiling pattern
formed when the sulfurs from the thiols bind to gold solid are
known. Therefore, Poisson statistics can be used to infer the
distance between surface immobilized ligands, and thus the
inter-binding-site distance on the target molecule, from the
concentrations of the thiols in solution. Further, the gold surface
itself and the attached SAM can be used as a scaffold to present
binding moieties, in a controlled, higher affinity geometry, to a
target molecule.
[0009] In a preferred embodiment, SAMs are generated that
incorporate two thiol types: 1) an inert tri-ethylene
glycol-terminated thiol; and 2) a nitrilo tri-acetic acid (NTA)
terminated thiol that when complexed with Ni, captures
histidine-tagged proteins or peptides. The density of NTA-thiol
within the SAM is varied to present varying densities of a
histidine-tagged binding moiety to a multi-valent target molecule.
The affinity of the interaction is plotted as a function of ligand
density within the SAM. A dramatic increase in the binding affinity
occurs at a critical surface density when the presented ligands are
close enough to each other to simultaneously bind to a common
target molecule. The solution concentrations of the two thiol types
and the dimensions of the tiling pattern that the thiols form on
the gold substrate are input into Poisson distribution equations to
extract the probable distance between binding sites on a target
molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the predicted structure of mixed self-assembled
monolayers made by doping a thiol solution with a nitriloacetic
acid terminated thiol.
[0011] FIG. 2 shows the results of the binding of hTBPc to
GST-2.times. peptide surfaces of differing densities.
[0012] FIG. 3 shows the binding of TBP target protein as a function
of peptide surface density.
[0013] FIG. 4 shows that the binding of hTBPc to surface
immobilized GST-2.times. is a non-linear function of the surface
density of the peptide.
[0014] FIG. 5 shows the three possible mechanistic models for
describing the interaction of TBP with reiterated peptide
activation motifs.
[0015] FIG. 6 shows titration curves summarizing competitive
inhibition experiments designed to measure the kinetics of
hTBPc-peptide activation motif binding.
[0016] FIG. 7 shows that TATA sequence DNA bound to hTBP does not
inhibit the interaction of hTBP with GST-4.times..
[0017] FIG. 8 shows that there is a synergistic increase in
affinity between hTBPc in solution and surface-bound GST-2.times.
when the density of immobilization is increased from 3.8% to
5.7%.
[0018] FIG. 9 shows competitive inhibition experiments
demonstrating that 2.times. ligands behave very differently in
solution versus when immobilized.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0019] Variable density nitrilotriacetic acid (NTA)-SAMs were used
to probe the binding site(s) of a biologically important molecule,
the human general transcription factor TATA box binding protein
(hTBP) [Burley, S. K. and Roeder, R. G. (1996) Biochemistry and
structural biology of transcription factor IID (TFIID). Annu. Rev.
Biochem. 65:769-799]. This transcription factor has been implicated
as a direct target of transcriptional activators such as VP16
[Ingles, J. C., M. Shales, W. D. Cress, S. J. Triezenberg and J.
Greenblatt. (1991) Reduced binding of TFIID to transcriptionally
compromised mutants of VP16. Nature. 351:588-590]. In fact, the
need for an activator is eliminated when TBP is artificially
tethered to a DNA promoter [Xiao, H., J. D. Friesen and J. T. Lis.
1995. Recruiting TATA-binding protein to a promoter:
transcriptional activation without an upstream activator. Mol. and
Cell. Biol. 15(10):5757-5761].
[0020] Transcriptional activator proteins are modular in that they
have functionally separable domains [Brent, R. and M. Ptashne.
(1985) A Eukaryotic transcriptional activator bearing the DNA
specificity of a prokaryotic repressor. Cell. 43:729-736], a DNA
binding domain, and an activating region. The structures of TBP
[Nikolov, D. B., H. Chen, E. D. Halay, A. A. Usheva, K. Hisatake,
D. K. Lee, R. G. Roeder and S. K. Burley. (1995) Crystal structure
of a TFIIB-TBP-TATA element ternary complex. Nature. 377:119-128]
and several activator DNA binding domains [Marmorstein, R., M.
Carey, M. Ptashne, and S. C. Harrison. 1992. DNA recognition by
Gal4: structure of a protein/DNA complex. Nature. 356:408-414;
Ellenberger et al., 1992; and Baleja, J. D., R. Marmorstein, S. C.
Harrison and G. Wagner. 1992]. The structure of the DNA-binding
domain of Cd2-Gal4 from Saccaromyces cervisiae in solution has been
solved, yet the structure of an activating region, alone or
complexed with a target molecule has remained elusive. Fundamental
questions as to how an activating region effects gene transcription
remain unanswered. One mechanistic model of gene activation
proposes that DNA-bound activators trigger transcription by merely
"recruiting" some necessary factor, perhaps TBP, to the promoter
through direct contact with the activating region [Triezenberg, S.
J. 1995. Structure and function of activation domains. Curr. Opin.
Genet. Dev. 5(2):190-196]. Another model proposes that activating
regions induce a conformational change in a target protein(s)
[Sheldon and Reinberg, 1995] or sequentially perform some function
until a threshold is reached which catalyzes gene
transcription.
[0021] In eukaryotes, more than one DNA-tethered activator is
typically required to achieve activated transcription and that
multiply bound activators transcribe synergistically [Lin, Y. S.,
M. Carey, M. Ptashne and M. R. Green. (1990) How different
eukaryotic transcriptional activators can cooperate promiscuously.
Nature 345:359-361]. Cryptic repeats of minimal activation motifs
have been identified in eukaryotic activators that, when tandemly
reiterated and tethered to DNA, efficiently activate transcription
in vitro [Blair et al., 1994; Tanaka, M. and W. Herr, (1994)
Reconstitution of transcriptional activation domains by reiteration
of short peptide segments reveals the modular organization of a
glutamine-rich activation domain. Mol. Cell. Biol.
14(9):6056-6067]. An eight amino acid minimal activation motif
(DFDLDMLG) derived from the prototypic mammalian activator VP16 was
recently identified [Tanaka, M. (1996) Modulation of promoter
occupancy by cooperative DNA binding and activation-function is a
major determinant of transcriptional regulation by activators in
vivo. Proc. Natl. Acad Sci. USA. 93(9):4311-4315]. As an exemplary
embodiment, this invention describes novel biophysical methods to
quantitate the kinetics, as well as investigate the mechanism, of
the interaction between hTBP and tandem repeats of the VP 16
minimal motif.
[0022] The interactions were characterized by SPR in a BIAcore
instrument. SPR is a fairly new optical technique for the real time
detection and kinetic analysis of intermolecular interactions
[Liedberg, B., C. Nylander and L. Lundstrom. (1983) Surface plasmon
resonance for gas detection and biosensing. Sens. Actuators.
4(2):299-304.; Daniels et al., 1988; Lofas, S. and Johnsson, B.
(1990) A novel hydrogel matrix on gold surfaces in surface plasmon
resonance sensors for fast and efficient covalent immobilization of
ligands. J Chem. Soc., Chem. Commun.: 1526-1528]. The basis of the
technology is as follows: ligands are immobilized on a surface;
putative target molecules are flowed over this surface; the protein
concentration at the solution-surface interface changes as target
binds ligand. The increased protein mass at the interface causes a
change in the optical properties of the system. The amount of new
protein recruited to the interfacial region can be quantitated by
measuring the change in the angle at which light reflected off the
interface is a minimum [for a review see Bamdad, C. 1997. Surface
plasmon resonance for measurements of biological interest. Current
Protocols in Molecular Biology 20.4.1-20.4.12.]. Changes in this
angle are measured in resonance units (RUs) where 1 RU is defined
as a change of {fraction (1/10,000)}th of a degree. A rule of thumb
is that for a distance of about 150 mn from the interface, 1 ng
protein/mm.sup.2 registers 10.sup.3 RUs.
[0023] SAMs were generated that incorporated an NTA group for the
specific binding of histidine-tagged peptides. The density of NTA
in the SAM was varied so that different amounts of a His-tagged
activation motif could be presented to TBP, in solution. SPR was
used to quantitate avidity effects between TBP and surface-bound
peptides as a function of peptide density.
[0024] FIG. 1 shows mixed self-assembled monolayers (SAMs) that
were generated by doping a thiol solution with an NTA-terminated
thiol and designed to capture histidine-tagged proteins. Sulfur
atoms deposit on gold substrates in a hexagonal tiling pattern 4.99
.ANG. on edge with three possible positions for thiol deposition
per hexagon. If it is assumed that in a well-ordered SAM all sites
are occupied, Poisson statistics can be used to calculate an
average distance between NTA-thiols for a given NTA concentration.
Equation (1) calculates how many hexagons must be filled before two
NTA-thiols are deposited. For a 3.8% NTA-thiol concentration in
solution, relative to EG.sub.3-thiol, an average of 17.5 hexagons
must be filled before 2 NTA ligands appear. For a 5.7% NTA
solution, 11.7 hexagons must be filled before an average of two NTA
ligands are deposited. The area of a hexagon 4.99 .ANG. on edge is
64.69 .ANG..sup.2 which is equal to the area of a square, 8.04
.ANG. on edge. NTA ligands on SAMs formed from a 3.8% NTA-thiol
solution would be an average of 29 .ANG. apart, while NTA ligands
in a SAM formed from a 5.7% NTA-thiol solution would be 23 .ANG.
apart. It was assumed that the concentration of NTA-thiol in
solution was equal to its concentration in the SAM; see FIG. 2 of
Sigal et al., 1996.
[0025] FIG. 2 shows that hTBPc in solution will not bind to
GST-2.times. peptide surfaces unless peptides are immobilized close
to one another. The BIAcore SPR instrument records changes in the
angle of minimum reflectance (RUs) as a function of time. Reagents
are flowed over individual flow cells of the SAM. The "square
waves" represent injections of protein "plugs" that interrupt the
constant buffer flow. An association constant can be derived from
an analysis of the initial phase of the injection and a
dissociation rate can be extracted from analysis of the system as
it returns to buffer flow. GST-2.times. or 4.times. fusion proteins
(X=DFDLDMLG) were separately immobilized on NTA-SAMs via
histidine-tags then hTBPc (124 nM) was injected over the surfaces.
An overlay of two SPR sensorgrams shows that hTBPc does not bind to
GST-2.times. immobilized on a 3.8% NTA-SAM (dashed line) but binds
very tightly when immobilized on a 5.7% NTA-SAM (solid line).
[0026] FIG. 3 shows the binding of target protein TBP measured by
SPR as a function of peptide surface density. A series of NTA-SAMs
were generated to display peptides at low to high density. When two
tandem repeats of the minimal activation peptide (GST2.times.) were
displayed at low density (1.3%-3.8%), human TBPc did not bind to
the surface. In contrast, a more dense GST-2.times. surface
(5.7%-11.4%), bound significant amounts of human TBPc. Fusion
proteins bearing four tandem repeats of the minimal activation
peptide (GST-4.times.) bound hTBPc whether the peptides were
displayed at low or high density. The stoichiometry of the
interaction was a constant, independent of the immobilization
density. Notably, at corresponding surface concentrations,
GST-2.times. bound half as much hTBPc as GST-4.times., suggesting
that two -2.times. modules immobilized at close proximity to each
other (high density) simultaneously contact one hTBPc molecule.
[0027] FIG. 4 shows that the binding of hTBPc to surface
immobilized GST-2.times. is a non-linear function of the surface
density of the peptide. Histidine-tagged peptides were separately
immobilized on SAMs presenting NTA over a wide range of surface
densities. SPR was used to quantitate the amount of target protein,
hTBPc that bound to each surface. The mass ratios of captured hTBPc
to surface immobilized peptide (GST-2.times. or -4.times.) was
plotted as a function of peptide concentration. The binding of
hTBPc to GST-4.times. (dashed line) is roughly constant over the
range of surface peptide densities. However, the binding of hTBPc
to GST-2.times. (solid line) approximates a step function of
GST-2.times. surface concentration.
[0028] FIG. 5 shows experiments that were designed to discriminate
between three possible mechanistic models to explain how reiterated
peptide activation motifs synergistically effect transcription of a
nearby gene. Model 1: two connected peptide motifs must be
positioned such that they can simultaneously bind to
quasi-identical sites on TBP. The bivalent, high affinity
interaction would keep the general transcription factor tethered
near the start site of transcription awaiting other steps in the
transcriptional activation process. Model 2: the binding of one or
two peptide activation motifs causes a conformational change in
TBP. The allosteric effect enhances the subsequent binding of
additional peptide motifs and a high affinity interaction results.
Model 3: a high affinity interaction occurs between the peptide
repeats and TBP but rather than resulting from a "bivalent"
interaction or an allosteric effect, it results from the simple
summation of multiple interactions between TBP and the entire
length of the activation peptide.
[0029] FIG. 6 shows titration curves, summarizing competitive
inhibition experiments, that yield IC.sub.50s that show the
4.times. peptide binds hTBPc 250 times tighter than the 2.times.
peptide. In order to quantitate the solution kinetics of hTBPc
binding to synthetic 4.times. peptides (4 tandem repeats of
DFDLDMLG) or 2.times. peptides (2 repeats), aliquots of hTBPc (124
nM) were incubated with increasing concentrations of either peptide
at 4.degree. C. for 1 hour. The mixtures were then separately
injected over identical SAMs that were pre-bound with GST-4.times..
Percent inhibition is plotted against the concentration of the
blocking peptide in solution. 0% inhibition was taken to be the
amount of hTBPc that bound to GST-4.times. surfaces when it was
incubated with buffer alone. Background levels of binding were
determined by injection of protein mixtures over naked GST
surfaces. An IC.sub.50 of 370 nM and 90 .mu.M describe the
equilibrium kinetics of hTBPc binding to 4.times. and 2.times.
peptides, respectively.
[0030] FIG. 7 shows that TATA sequence DNA bound to hTBP does not
inhibit the hTBP/GST-4.times. interaction. N-terminally
histidine-tagged hTBP was bound to NTA-SAMs and the mass of bound
protein was quantitated and recorded by a BIAcore SPR instrument.
The SAMs, bound with hTBP, were then removed from the instrument
and separately incubated at RT for 15 minutes with solutions
containing equal mass amounts of either DNA bearing the hTBP TATA
recognition sequence or random sequence DNA (150 MM NaCl; 50
nanomoles DNA). The SAMs were then washed in running buffer and
re-docked in the SPR instrument. The increase in absolute RUs of
the baseline indicated that the TATA sequence DNA bound to surface
immobilized hTBP with roughly 1:1 stoichiometry while the random
DNA bound only nonspecifically. Protein plugs of GST4.times. were
separately injected over these surfaces; the presence of DNA, bound
nonspecifically or specifically, was not inhibitory to the
subsequent binding of GST-4.times. to hTBP. Additionally, the
measured association and dissociation rates, which were not
affected by DNA-binding, were identical to those measured with
GST-4.times. bound to the SAM and TBP in solution.
[0031] FIG. 8 shows that there is a synergistic increase in
affinity between hTBPc in solution and surface-bound GST-2.times.
when the density of immobilization is increased from 3.8% to 5.7%.
Low (3.8% NTA) then high (5.7% NTA) density SAMs were docked in an
SPR device. Histidine-tagged GST-2.times. and GST-4.times. fusion
proteins (0.3 mg/ml) were separately immobilized on individual flow
cells of the SAMs. The mass of the immobilized species is recorded
in resonance units (RUs), where 1000 RUs=1 ng protein/mm.sup.2. One
RU results from a net change of {fraction (1/10,000)} of a degree
in the angle of minimum reflectance off of the differential
dielectric interface of the sensing wave. hTBPc (124 nM) was then
injected over the derivatized surfaces. The mass of the captured
analyte was obtained by taking the difference between RUs recorded
10 seconds prior to and 25 seconds after the injection. When
GST-2.times. was immobilized at low density it was not able to bind
hTBP. However, when immobilized at slightly higher density, a high
affinity interaction resulted. The stoichiometry of surface
immobilized GST-4.times. binding to hTBPc was relatively constant
but, notably, twice that of GST-2.times. binding to hTBPc which
reinforces the idea that two -2.times. ligands bind one hTBPc
molecule.
[0032] FIG. 9 shows competitive inhibition experiments in which
2.times. ligands behave very differently in solution than when
surface immobilized and that reiterated minimal activation motifs
effectively compete for the same binding site(s) on hTBP as the
parent protein. Histidine-tagged GST-4.times. or GST-2.times. were
separately immobilized on NTA-SAMs docked in a BIAcore SPR
instrument. hTBPc(residues 155-335) or hTBP (full length) was
pre-incubated at high concentration (35 .mu.M) with either buffer,
a synthetic 2.times. peptide (X=DFDLDMLG) at 1:4 stoichiometry, a
4.times. peptide at 1:2 stoichiometry, or a 1X-linker-1X peptide
DFDLDMLG-((Ser).sub.4Gly.sub.1).sub.3-DFDLDMLG) at 1:2
stoichiometry for 1 h at 40.degree. C. Just prior to injection over
the derivatized surfaces, the pre-incubation mixtures were diluted
such that the final hTBP concentration was (124 nM). The synthetic
4.times. and 1X-linker-1X peptides blocked the interaction of hTBP
with surface immobilized ligands but 2.times. peptides were not
inhibitory. Histidine-tagged Gal4(1147)+VP16(413-490) were
similarly immobilized on NTA-SAMs. hTBP was preincubated, as
described above, with either buffer or 4.times. peptide then
diluted and injected over the VP16 presenting surfaces. The 32
amino acid 4.times. peptide effectively blocked the interaction of
hTBP with the 78 amino acid VP16 activation domain.
[0033] A panel of variable density NTA-SAMs were prepared by
diluting the concentration of the active component, NTA-thiol,
relative to that of the inert component, EG.sub.3-thiol, in ethanol
solutions. Gold-coated glass slides were incubated in solutions
containing 1.3%, 3.8%, 5.7%, or 11.4% NTA-thiol, with the total
thiol concentration constant at 1 mM. The SAMs were glued onto
blank CM-5 SPR chip cassettes and docked into a BIAcore instrument.
A 16-mer peptide comprised of two repeats of the eight amino acid
minimal activation motif (X=DFDLDMLG), derived from the human
activator VP16, was fused to histidine-tagged GST (GST-2.times.).
The fusion proteins were then immobilized on variable density SAMs
through complexation of the NTA group by the protein's histidine
tag. This generated a series of surfaces that displayed peptides at
incrementally decreasing distances from each other. The core region
of human TBP (hTBPc: residues 155-335) (Nikolov et al., 1995) was
injected over the peptide surfaces. GST-2.times. immobilized at low
density (1.3%-3.8%), was unable to bind hTBPc. In contrast, when
the same concentration hTBPc was injected over a more dense
(5.7%-11.4%) GST-2.times. surface, where the average distance
between peptide motifs would be smaller, a high affinity
interaction resulted (see FIGS. 2 and 8). As a control, fusion
proteins bearing four iterations of the minimal motif
(GST-4.times.) were immobilized on the different density SAMs and
assayed for the ability to bind the target molecule. Human TBPc, in
solution, bound identically to GST-4.times. surfaces irrespective
of the peptide density (see FIG. 3 and FIG. 8).
[0034] As the graph of FIG. 4 shows, the stoichiometry of hTBPc
binding to GST-4.times. derivatized surfaces is a constant,
independent of the immobilization density. In contrast, the binding
of hTBPc to GST-2.times. surfaces is a non-linear function of the
surface density. Notably, at corresponding surface concentrations,
GST-2.times. bound half as much hTBPc as GST-4.times., suggesting
that two 2.times. modules immobilized at close proximity to each
other (high density) simultaneously contact one hTBPc molecule.
Kinetic rate constants were extracted by analyzing association and
dissociation phases of sensorgram curves using a non-linear
regression curve fitting program: BIAevaluation, version 2.1. The
analysis assumed pseudo-first order reactions. The interaction
between GST4.times. and hTBPc was characterized by an average
association rate of 2.5.times.10.sup.4 s.sup.-1 M.sup.-1 and an
average dissociation rate of 4.times.10.sup.-4 s.sup.-1, yielding a
calculated average k.sub.d of 16.times.10.sup.-9 M. Standard errors
obtained for each SPR experiment were considerably smaller than the
variation in kinetic rates measured among several experiments using
a wide range of NTA concentrations. There could be as much as a
two-fold variation in the calculated k.sub.d. Sensorgram
association curves from the binding of hTBPc to GST-2.times. could
not be fit by pseudo first order kinetics, again consistent with
the idea that two -2.times. modules bind one hTBPc molecule.
However, the dissociation phase of the sensorgram was well fit and
yielded an average k.sub.d of 1.5.times.10.sup.-3+/-0.13 s.sup.-1
for the interaction. The almost ten-fold difference between the
4.times. k.sub.d and 2.times. k.sub.d may indicate that the
2.times. dissociation curve is the superposition of two decay
rates, corresponding to two dissociating species.
[0035] Note that at high NTA density, the chip surface acted as a
rigid linker between two -2.times. modules to mimic a 4.times.
module, thus creating a higher affinity ligand. Three possible
models might explain why the 4.times. peptide is a higher affinity
ligand for hTBPc than a 2.times. peptide (See FIG. 5). Model 1
proposes that the 4.times. peptide is a "bivalent" ligand that
simultaneously and cooperatively binds more than one site on the
target protein, producing a high affinity interaction characterized
by a slower off-rate (Jencks, W. P. 1981. On the attribution and
additivity of binding energies. Proc. Natl. Acad Sci. USA.
78(7):4046-4050.). Model 2 says the binding of one recognition
motif causes an allosteric effect that enhances the binding of
subsequent motifs. Four connected minimal motifs provide for an
increased local concentration of ligand available for the second
higher affinity interaction. Model 3 proposes that the higher
affinity interaction is the result of the summation of multiple
interactions of equal strength between the target protein and the
entire length of the peptide. A prediction of Model 1 is that
2.times. peptides, free in solution, will interact with hTBPc
independently and exhibit a faster off-rate which is characteristic
of monovalent binding. Therefore, if hTBPc is pre-bound by peptide
in solution, the 4.times. peptide should be a much better inhibitor
of hTBPc binding to surface immobilized ligand than the 2.times.
peptide. Model 2 predicts that hTBPc pre-bound by 4.times. or
2.times. peptides (at twice the concentration) would be similarly
inhibited, so long as incubation concentrations were high enough to
compensate for the 4.times. local concentration advantage. Model 3
implies that mutation of amino acids within the peptide would
decrease its affinity for TBP as an approximately linear function
of the number of mutations.
[0036] In order to compare dissociation rates, aliquots of hTBPc
were pre-incubated at very high concentration (35 .mu.M) with
either buffer, 2.times. peptide (1:4 stoichiometry), or 4.times.
peptide (1:2 stoichiometry), then diluted to the usual hTBPc
concentration (124 nM) before injection over GST-4.times. surfaces.
Synthetic 2.times. (16-mer) and 4.times. (32-mer) peptides were
used to eliminate possible interference from GST. FIG. 9 shows that
the preincubation of hTBPc with 2.times. peptide was in no way
inhibitory to its interaction with surface immobilized
GST-4.times.. In contrast, preincubation of hTBPc with 4.times.
peptide completely abolished the interaction. Additional
experiments showed that the 32-mer, but not the 16-mer peptide,
also blocked the binding of hTBPc to high density GST-2.times.
surfaces, again demonstrating that GST-2.times., immobilized at
high density, behaves like GST-4.times..
[0037] The experiments tabulated in FIG. 9 argue against the
allosteric effect model but are consistent with Models 1 and 3. The
question is, does the increased binding energy of the hTBP-4.times.
interaction result from the cumulative effect of multiple bonds
along the length of the peptide or from the synergistic effect of
two minimal motifs simultaneously binding to the target molecule,
with the intervening amino acids merely serving as a tether between
the two? A synthetic 31 amino acid peptide consisting of two
minimal motifs (DFDLDMLG) separated by a flexible linker
((Ser.sub.4 Glyl).sub.3) was generated. This peptide, 1X-linker-1X,
when preincubated with hTBP (under the same conditions described
above) inhibited by 83% the complex's ability to bind to surface
immobilized GST-4.times. (see FIG. 9). These results reinforce the
premise of Model 1 and imply that the enhanced strength of binding
between hTBP and the 4.times. peptide is due to a synergistic
effect caused by two connected minimal activation motifs
simultaneously binding to two separate and discrete sites on hTBP.
One may also infer, from the last experiment, that the interaction
between minimal activation motifs and hTBP is specific.
[0038] Next the kinetics of the surface interaction to analogous
interactions in solution were compared. A series of equilibrium
inhibition experiments were performed to characterize the solution
interactions between hTBPc and 2.times. or 4.times. peptides.
Aliquots of hTBPc, (124 nM), were mixed with increasing amounts of
synthetic 2.times. or 4.times. peptide then incubated at 4.degree.
for 1 hour prior to injection over GST-4.times. surfaces. Titration
curves (see FIG. 6) yield an IC.sub.50 of 370 nM for the 4.times.
peptide and 90 .mu.M for the 2.times. peptide binding to hTBPc. In
summary, the 4.times. peptide binds hTBPc about 250-times better
than the 2.times. peptide. This is the relative difference between
monovalent and bivalent binding of hTBPc. The interaction between
the 4.times. peptide and hTBPc in solution is about 20-times weaker
than the comparable surface interaction where diffusion is
limited.
[0039] The physiological relevance of the interaction between hTBP
and the reiterated minimal motifs was investigated. It has been
argued that the widely observed in vitro interactions between TBP
and activation domains are artifacts resulting from a nonspecific
interaction between TBP's basic DNA-binding region and the acidic
peptides. To rule out this possibility, N-terminally
histidine-tagged hTBP was immobilized on NTA-SAMs then separately
incubated with either: a) TATA sequence DNA; or b) DNA that did not
contain a hTBP recognition sequence. GST-4.times. was then injected
over the derivatized surfaces. DNA that did not contain a TATA
sequence did not bind to the immobilized hTBP significantly. DNA
containing a TATA sequence bound to immobilized hTBP with
approximate 1:1 stoichiometry but was in no way inhibitory to the
subsequent binding of GST-4.times. (see FIG. 7). In fact, hTBPc
complexed by its cognate DNA bound roughly twice as much
GST-4.times. as the uncomplexed hTBPc. This result is consistent
with the observation that hTBPc exists as a dimer that is disrupted
upon DNA binding (Taggart, A. K. P. and B. F. Pugh. 1996.
Dimerization of TFIID when not bound to DNA. Science.
272:1331-1333.). The binding of an activating region does not seem
to disrupt hTBPc dimerization.
[0040] A competitive inhibition experiment was performed to
determine whether the 4.times. peptide could block the interaction
between hTBP and the native activation domain of VP16. A
histidine-tagged Gal4(1-147)+VP16(413-490) fusion protein was
immobilized on NTA-SAMs. hTBP was incubated with buffer or 4.times.
peptide then injected over VP16 derviatized surfaces. The last two
lines of FIG. 9 show that preincubation of hTBP with the 4.times.
peptide (32 amino acids) completely abolished the hTBP-VP 16 (78
amino acids) interaction. This result is consistent with the idea
that minimal activation motifs recognize the same binding site(s)
on hTBP as the parent activator.
[0041] In conclusion, SAMs were used to form biospecific rigid,
nano-scale probe arrays of known surface density and then utilized
to determine the number of binding sites on a target molecule and
an approximate distance between sites. This approach is not
hampered by the vagaries of secondary or tertiary structures that
would be encountered by using DNA or peptide spacers to determine
distances between active sites. SPR was used to show that the
avidity between TBP, in solution, and surface immobilized peptides
was a non-linear function of peptide surface density.
[0042] Peptides immobilized on a 3.8% NTA-SAM were not able to bind
hTBP, while peptides presented on a 5.7% NTA-SAM bound TBP with
nano-molar affinity. The findings are consistent with the idea that
this large increase in binding strength marks the transition
between mono- and bivalent binding of the target protein.
Individual 8 amino acid minimal activation motifs separated by a 15
amino acid flexible linker bound hTBP nearly as well as four tandem
repeats of the motif, leading to the conclusion that hTBP has at
least two discrete sites capable of simultaneously interacting with
the 8 amino acid motif. Calculations based on an assumed Poisson
distribution of NTA in the SAM indicate that the surfaces that did
not bind hTBP (3.7% NTA) presented peptides an average distance of
29 .ANG. apart while peptides in denser arrays (5.7% NTA) that
bound hTBP with high avidity were on average 2 .ANG. apart.
[0043] The crystal structure of hTBPc has been solved (Nikolov et
al., 1995). The peptide consists of two imperfect repeats that form
a two-domain saddle shaped DNA-binding protein with two-fold
intramolecular symmetry. TBP binds DNA with the concave underside
of its "saddle" shape. The general transcription factor TFIIB binds
near the TBP/DNA complex at the downstream end leaving the convex
"seat" of the saddle available for other intermolecular
interactions. Quasi-identical structures composed of basic helices
and P sheets flank the seat of the saddle. Mirror image helices H2
and H2' are separated by distances on the order of 20 .ANG.. It is
conceivable that the minimal activation motifs, described herein,
simultaneously bind to two-fold related pseudo-identical
recognition sites that may be separated by approximately 23
.ANG..
[0044] Similar schemes can be devised to determine distances
between active sites on other bivalent molecules or complexes. Of
particular interest are dimeric hormone receptors whose signaling
activity depends on its association state. Detailed knowledge of
distances between active sites would allow for the rational design
of agonist or antagonist drugs.
Experimental Methods
[0045] Protein preparation: hTBPc was prepared according to Nikolov
et al., 1996 and full length histidine-tagged hTBP according to Lee
et al. [Lee, W. S., C. C. Kao, G. O. Bryant, X. Liu and A. J. Berk.
(1991) Adenovirus EIA activation domain binds the basic repeat in
the TATA box transcription factor. Cell 67:365-376]. Glutathione
S-transferase (GST) fusion proteins were prepared according to
Tanaka, 1996. The preparation of Gal4-VP 16 is described by Hori,
R., S. Pyo and M. Carey, 1995. Protease footprinting reveals a
surface on transcription factor TFHB that serves as an interface
for activators and co-activators. Proc. Natl. Acad Sci. USA.
92(13):6047-6051.
[0046] DNA: TATA sequence DNA was prepared according to Parvin et
al. [Parvin, J. D., R. J. McCormick, P. A. Sharp, and D. E. Fisher.
1995. Pre-bending of a promoter sequence enhances affinity for the
TATA-binding factor. Nature. 373:724-727] with the exception that
it was not circularized. A 50 base-pair double stranded oligo
containing 2 Gal4 binding sites, synthesized and quantitated by
GibcoBRL, Life Technologies Inc., Grand Island, N.Y., was used as
non-specific control DNA. Equal mass amounts of specific vs.
non-specific DNA were added.
[0047] Synthetic peptides: Peptides were generated by F-MOC
synthesis and quantitated by amino acid analysis, analytical HPLC
and mass spectroscopy.
[0048] The preparation self-assembled monolayers: NTA-SAMs were
prepared according to Sigal et al., 1996. A panel of incrementally
different density NTA surfaces was generated by serial dilution of
a stock solution containing 11.4% NTA-thiol, relative to
tri-ethylene glycol terminated thiol, into solutions containing the
tri-ethylene glycol terminated thiol alone. Total thiol
concentration was kept constant at 1 mM. NTA-SAMs were stored under
argon for up to 1 week prior to use. Background levels of binding
were assessed by passing reactants over underivatized GST surfaces
and subtracted.
[0049] Surface plasmon resonance: Experiments were carried out in a
BIAcore instrument at room temperature in phosphate buffered saline
(PBS) (137 mM NaCl) running at a constant flow rate of 5 .mu.l/min.
Sample injection volumes (plugs) were 35 .parallel.l. Association
and dissociation rate constants were extracted from the data with
BIAevaluation software, version 2.1, assuming a pseudo first order
kinetics model: A+BAB. Error rates were taken from the deviation of
measurements among multiple experiments performed on surfaces of
different NTA densities with a range of protein concentrations and
using several different protein preparations, of the same species,
to account for variation of the active concentration of a
component.
[0050] Statistical calculations: Sulfur atoms bind to gold to form
a face-centered hexagonal tiling pattern 4.99 .ANG. on edge. In an
ordered monolayer, all the positions of the hexagon are occupied by
a thiol. Each vertex is shared by three hexagons, so there are
three possible positions for thiol deposition per hexagon. If the
thiol solution is doped with a derivatized species of thiol, such
as ours is, the average number of NTA-thiols deposited per some
number of hexagons (.lambda.), can be calculated, assuming Poisson
statistics, for a given NTA-thiol concentration. (It was assumed
that the concentration of NTA-thiol in solution was equal to its
concentration in the SAM; see FIG. 2 of Sigal et al., 1996).
Equation (1) of FIG. 1 calculates how many hexagons, on average,
must be filled before two NTA-thiols are deposited. For a 3.8%
NTA-thiol concentration in solution, relative to EG.sub.3-thiol, an
average of 17.5 hexagons must be filled before 2 NTA ligands
appear. For a 5.7% NTA solution, 11.7 hexagons must be filled
before an average of two NTA ligands are deposited. The area of a
hexagon 4.99 .ANG. on edge is 64.69 .ANG..sup.2 which is equal to
the area of a square, 8.04 .ANG. on edge. 17.5 hexagons would
occupy the same area as a square (17.5.times.8.04.sup.2).sup.1/2
.ANG. on edge, which equals 33.6 .ANG.. Two NTA ligands were
arbitrarily placed in a square representing 17.5 hexagons either
33.6 .ANG. or 23.8 .ANG. apart (See FIG. 1).
[0051] Since there are equal numbers of nearest and next-nearest
neighbors, the average of these two distances is a first order
approximation of the average distance between ligands resulting
from a random distribution. According to this model, NTA ligands on
SAMs formed from a 3.8% NTA-thiol solution would be an average of
29 .ANG. apart, while NTA ligands in a SAM formed from a 5.7%
NTA-thiol solution would be 23 .ANG. apart. Calculations were done
to evaluate the contribution of clustering using Poisson
statistics.
[0052] Equation 2 calculates the probability, P, of having n NTA
ligands per unit area, where .lambda., equals the average number of
NTAs per unit area. Equation 3 calculates the ratio of the
probabilities of having one NTA ligand to two NTA ligands deposited
per unit area. It is 17-times more likely to get one NTA than two,
per unit area, for 3.8% NTA-thiol SAMs and 11 times more likely at
5.7% NTA concentration.
P(n)=e.sup.-.lambda..lambda..sup.n/n! (2) 1 P ( 1 ) P ( 2 ) = - ( 3
sites ) ( [ 0.038 ] NTA ) [ ( 3 ) ( 0.038 ) ] 1 / 1 ! - ( 3 sites )
( [ 0.038 ] NTA ) [ ( 3 ) ( 0.038 ) ] 2 / 2 ! ( 3 )
[0053] All publications cited in this application are hereby
incorporated by reference. The citation of any publication is for
its disclosure prior to the filing date and should not be construed
as an admission that the present invention is not entitled to
antedate such publication by virtue of prior invention.
[0054] It is to be understood that the above invention is not
limited to the particular embodiments described which are meant to
be for illustrative purposes only. Variations and modifications of
these embodiments may be made that are still included in the
description of this invention and fall within the scope of the
appended claims.
Sequence CWU 1
1
20 1 1876 DNA Homo sapiens CDS (242)...(1261) hTBP 1 cgcggccgcg
gttcgctgtg gcgggcgcct gggccgccgg ctgtttaact tcgcttccgc 60
tggcccatag tgatctttgc agtgacccag cagcatcact gtttcttggc gtgtgaagat
120 aacccaagga attgaggaag ttgctgagaa gagtgtgctg gagatgctct
aggaaaaaat 180 tgaatagtga gacgagttcc agcgcaaggg tttctggttt
gccaagaaga aagtgaacat 240 c atg gat cag aac aac agc ctg cca cct tac
gct cag ggc ttg gcc tcc 289 Met Asp Gln Asn Asn Ser Leu Pro Pro Tyr
Ala Gln Gly Leu Ala Ser 1 5 10 15 cct cag ggt gcc atg act ccc gga
atc cct atc ttt agt cca atg atg 337 Pro Gln Gly Ala Met Thr Pro Gly
Ile Pro Ile Phe Ser Pro Met Met 20 25 30 cct tat ggc act gga ctg
acc cca cag cct att cag aac acc aat agt 385 Pro Tyr Gly Thr Gly Leu
Thr Pro Gln Pro Ile Gln Asn Thr Asn Ser 35 40 45 ctg tct att ttg
gaa gag caa caa agg cag cag cag caa caa caa cag 433 Leu Ser Ile Leu
Glu Glu Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln 50 55 60 cag cag
cag cag cag cag cag cag caa cag caa cag cag cag cag cag 481 Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80
cag cag cag cag cag cag cag cag cag cag cag cag cag caa cag gca 529
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala 85
90 95 gtg gca gct gca gcc gtt cag cag tca acg tcc cag cag gca aca
cag 577 Val Ala Ala Ala Ala Val Gln Gln Ser Thr Ser Gln Gln Ala Thr
Gln 100 105 110 gga acc tca ggc cag gca cca cag ctc ttc cac tca cag
act ctc aca 625 Gly Thr Ser Gly Gln Ala Pro Gln Leu Phe His Ser Gln
Thr Leu Thr 115 120 125 act gca ccc ttg ccg ggc acc act cca ctg tat
ccc tcc ccc atg act 673 Thr Ala Pro Leu Pro Gly Thr Thr Pro Leu Tyr
Pro Ser Pro Met Thr 130 135 140 ccc atg acc ccc atc act cct gcc acg
cca gct tcg gag agt tct ggg 721 Pro Met Thr Pro Ile Thr Pro Ala Thr
Pro Ala Ser Glu Ser Ser Gly 145 150 155 160 att gta ccg cag ctg caa
aat att gta tcc aca gtg aat ctt ggt tgt 769 Ile Val Pro Gln Leu Gln
Asn Ile Val Ser Thr Val Asn Leu Gly Cys 165 170 175 aaa ctt gac cta
aag acc att gca ctt cgt gcc cga aac gcc gaa tat 817 Lys Leu Asp Leu
Lys Thr Ile Ala Leu Arg Ala Arg Asn Ala Glu Tyr 180 185 190 aat ccc
aag cgg ttt gct gcg gta atc atg agg ata aga gag cca cga 865 Asn Pro
Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Arg 195 200 205
acc acg gca ctg att ttc agt tct ggg aaa atg gtg tgc aca gga gcc 913
Thr Thr Ala Leu Ile Phe Ser Ser Gly Lys Met Val Cys Thr Gly Ala 210
215 220 aag agt gaa gaa cag tcc aga ctg gca gca aga aaa tat gct aga
gtt 961 Lys Ser Glu Glu Gln Ser Arg Leu Ala Ala Arg Lys Tyr Ala Arg
Val 225 230 235 240 gta cag aag ttg ggt ttt cca gct aag ttc ttg gac
ttc aag att cag 1009 Val Gln Lys Leu Gly Phe Pro Ala Lys Phe Leu
Asp Phe Lys Ile Gln 245 250 255 aac atg gtg ggg agc tgt gat gtg aag
ttt cct ata agg tta gaa ggc 1057 Asn Met Val Gly Ser Cys Asp Val
Lys Phe Pro Ile Arg Leu Glu Gly 260 265 270 ctt gtg ctc acc cac caa
caa ttt agt agt tat gag cca gag tta ttt 1105 Leu Val Leu Thr His
Gln Gln Phe Ser Ser Tyr Glu Pro Glu Leu Phe 275 280 285 cct ggt tta
atc tac aga atg atc aaa ccc aga att gtt ctc ctt att 1153 Pro Gly
Leu Ile Tyr Arg Met Ile Lys Pro Arg Ile Val Leu Leu Ile 290 295 300
ttt gtt tct gga aaa gtt gta tta aca ggt gct aaa gtc aga gca gaa
1201 Phe Val Ser Gly Lys Val Val Leu Thr Gly Ala Lys Val Arg Ala
Glu 305 310 315 320 att tat gaa gca ttt gaa aac atc tac cct att cta
aag gga ttc agg 1249 Ile Tyr Glu Ala Phe Glu Asn Ile Tyr Pro Ile
Leu Lys Gly Phe Arg 325 330 335 aag acg acg taa tggctctcat
gtacccttgc ctcccccacc cccttctttt 1301 Lys Thr Thr * ttttttttta
aacaaatcag tttgttttgg tacctttaaa tggtggtgtt gtgagaagat 1361
ggatgttgag ttgcagggtg tggcaccagg tgatgccctt ctgtaagtgc ccaccgcggg
1421 atgccgggaa ggggcattat ttgtgcactg agaacaccgc gcagcgtgac
tgtgagttgc 1481 tcataccgtg ctgctatctg ggcagcgctg cccatttatt
tatatgtaga ttttaaacac 1541 tgctgttgac aagttggttt gagggagaaa
actttaagtg ttaaagccac ctctataatt 1601 gattggactt tttaatttta
atgtttttcc ccatgaacca cagtttttat atttctacca 1661 gaaaagtaaa
aatctttttt aaaagtgttg tttttctaat ttataactcc taggggttat 1721
ttctgtgcca gacacattcc acctctccag tattgcagga cggaatatat gtgttaatga
1781 aaatgaatgg ctgtacatat ttttttcttt cttcagagta ctctgtacaa
taaatgcagt 1841 ttataaaagt gttaaaaaaa aaaaaaaaaa aaaaa 1876 2 339
PRT Homo sapiens 2 Met Asp Gln Asn Asn Ser Leu Pro Pro Tyr Ala Gln
Gly Leu Ala Ser 1 5 10 15 Pro Gln Gly Ala Met Thr Pro Gly Ile Pro
Ile Phe Ser Pro Met Met 20 25 30 Pro Tyr Gly Thr Gly Leu Thr Pro
Gln Pro Ile Gln Asn Thr Asn Ser 35 40 45 Leu Ser Ile Leu Glu Glu
Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala 85 90 95
Val Ala Ala Ala Ala Val Gln Gln Ser Thr Ser Gln Gln Ala Thr Gln 100
105 110 Gly Thr Ser Gly Gln Ala Pro Gln Leu Phe His Ser Gln Thr Leu
Thr 115 120 125 Thr Ala Pro Leu Pro Gly Thr Thr Pro Leu Tyr Pro Ser
Pro Met Thr 130 135 140 Pro Met Thr Pro Ile Thr Pro Ala Thr Pro Ala
Ser Glu Ser Ser Gly 145 150 155 160 Ile Val Pro Gln Leu Gln Asn Ile
Val Ser Thr Val Asn Leu Gly Cys 165 170 175 Lys Leu Asp Leu Lys Thr
Ile Ala Leu Arg Ala Arg Asn Ala Glu Tyr 180 185 190 Asn Pro Lys Arg
Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Arg 195 200 205 Thr Thr
Ala Leu Ile Phe Ser Ser Gly Lys Met Val Cys Thr Gly Ala 210 215 220
Lys Ser Glu Glu Gln Ser Arg Leu Ala Ala Arg Lys Tyr Ala Arg Val 225
230 235 240 Val Gln Lys Leu Gly Phe Pro Ala Lys Phe Leu Asp Phe Lys
Ile Gln 245 250 255 Asn Met Val Gly Ser Cys Asp Val Lys Phe Pro Ile
Arg Leu Glu Gly 260 265 270 Leu Val Leu Thr His Gln Gln Phe Ser Ser
Tyr Glu Pro Glu Leu Phe 275 280 285 Pro Gly Leu Ile Tyr Arg Met Ile
Lys Pro Arg Ile Val Leu Leu Ile 290 295 300 Phe Val Ser Gly Lys Val
Val Leu Thr Gly Ala Lys Val Arg Ala Glu 305 310 315 320 Ile Tyr Glu
Ala Phe Glu Asn Ile Tyr Pro Ile Leu Lys Gly Phe Arg 325 330 335 Lys
Thr Thr 3 181 PRT Homo sapiens PEPTIDE (1)...(181) hTBPc 3 Ala Ser
Glu Ser Ser Gly Ile Val Pro Gln Leu Gln Asn Ile Val Ser 1 5 10 15
Thr Val Asn Leu Gly Cys Lys Leu Asp Leu Lys Thr Ile Ala Leu Arg 20
25 30 Ala Arg Asn Ala Glu Tyr Asn Pro Lys Arg Phe Ala Ala Val Ile
Met 35 40 45 Arg Ile Arg Glu Pro Arg Thr Thr Ala Leu Ile Phe Ser
Ser Gly Lys 50 55 60 Met Val Cys Thr Gly Ala Lys Ser Glu Glu Gln
Ser Arg Leu Ala Ala 65 70 75 80 Arg Lys Tyr Ala Arg Val Val Gln Lys
Leu Gly Phe Pro Ala Lys Phe 85 90 95 Leu Asp Phe Lys Ile Gln Asn
Met Val Gly Ser Cys Asp Val Lys Phe 100 105 110 Pro Ile Arg Leu Glu
Gly Leu Val Leu Thr His Gln Gln Phe Ser Ser 115 120 125 Tyr Glu Pro
Glu Leu Phe Pro Gly Leu Ile Tyr Arg Met Ile Lys Pro 130 135 140 Arg
Ile Val Leu Leu Ile Phe Val Ser Gly Lys Val Val Leu Thr Gly 145 150
155 160 Ala Lys Val Arg Ala Glu Ile Tyr Glu Ala Phe Glu Asn Ile Tyr
Pro 165 170 175 Ile Leu Lys Gly Phe 180 4 2211 DNA Herpes simplex
virus type 2 CDS (88)...(1560) VP16 4 ggatccctcc ccccctctcc
gccgccgggc gctcgggcac gtctcattcg cctctcgaga 60 tcgttattcc
cggacccaac cgccccc atg gac ctg ttg gtc gac gat ctg ttt 114 Met Asp
Leu Leu Val Asp Asp Leu Phe 1 5 gcg gac cgg gac ggg gtt tcg cca ccg
ccc ccc agg cca gcc ggg ggt 162 Ala Asp Arg Asp Gly Val Ser Pro Pro
Pro Pro Arg Pro Ala Gly Gly 10 15 20 25 ccc aag aac acc cca gcc gcc
cct ccg ctg tac gcc acc ggt cgg ctg 210 Pro Lys Asn Thr Pro Ala Ala
Pro Pro Leu Tyr Ala Thr Gly Arg Leu 30 35 40 agt cag gcc cag ctg
atg ccc tcg ccg ccc atg ccc gtc ccc ccc gcg 258 Ser Gln Ala Gln Leu
Met Pro Ser Pro Pro Met Pro Val Pro Pro Ala 45 50 55 gcc ctg ttt
aac cgt ctc ctc gac gat ctg ggc ttc agc gcg ggt ccc 306 Ala Leu Phe
Asn Arg Leu Leu Asp Asp Leu Gly Phe Ser Ala Gly Pro 60 65 70 gcg
ctg tgt acc atg cta gat acc tgg aac gag gac ctg ttc tct ggg 354 Ala
Leu Cys Thr Met Leu Asp Thr Trp Asn Glu Asp Leu Phe Ser Gly 75 80
85 ttc ccg acc aac gcc gac atg tac cgg gag tgc aag ttt ctg tcg acg
402 Phe Pro Thr Asn Ala Asp Met Tyr Arg Glu Cys Lys Phe Leu Ser Thr
90 95 100 105 ctg ccc agc gac gtg atc gac tgg ggg gat gcg cac gtc
ccc gag cgc 450 Leu Pro Ser Asp Val Ile Asp Trp Gly Asp Ala His Val
Pro Glu Arg 110 115 120 tcc ccg atc gac att cgc gcc cac ggc gac gtg
gcg ttc ccc acc ctg 498 Ser Pro Ile Asp Ile Arg Ala His Gly Asp Val
Ala Phe Pro Thr Leu 125 130 135 ccc gcc acc cgc gac gag ctg cct tcg
tac tac gag gcc atg gcg cag 546 Pro Ala Thr Arg Asp Glu Leu Pro Ser
Tyr Tyr Glu Ala Met Ala Gln 140 145 150 ttt ttc cgc ggt gag ctg cgg
gcg cgg gag gag agc tac cgg acc gtg 594 Phe Phe Arg Gly Glu Leu Arg
Ala Arg Glu Glu Ser Tyr Arg Thr Val 155 160 165 ttg gca aat ttt tgc
tcg gcc ctg tac cgg tac ctg cgc gcc agc gtt 642 Leu Ala Asn Phe Cys
Ser Ala Leu Tyr Arg Tyr Leu Arg Ala Ser Val 170 175 180 185 cgg cag
cta cac cgc cag gca cac atg cgg ggc cgc aac cgc gac ctg 690 Arg Gln
Leu His Arg Gln Ala His Met Arg Gly Arg Asn Arg Asp Leu 190 195 200
cgg gag atg ctg cgc acc acg atc gcg gac agg tac tac cgc gag acc 738
Arg Glu Met Leu Arg Thr Thr Ile Ala Asp Arg Tyr Tyr Arg Glu Thr 205
210 215 gcg cgc ctg gcg cgc gtc ctg ttt ctg cat cta tac ctc ttt ctg
agc 786 Ala Arg Leu Ala Arg Val Leu Phe Leu His Leu Tyr Leu Phe Leu
Ser 220 225 230 cgc gag atc cta tgg gcc gcg tac gcc gag cag atg atg
cgg ccc gat 834 Arg Glu Ile Leu Trp Ala Ala Tyr Ala Glu Gln Met Met
Arg Pro Asp 235 240 245 ctg ttc gac ggc ctc tgc tgc gac ctg gag agc
tgg cgc cag ttg gcg 882 Leu Phe Asp Gly Leu Cys Cys Asp Leu Glu Ser
Trp Arg Gln Leu Ala 250 255 260 265 tgt ctg ttt cag ccc ctg atg ttt
atc aac gga tcg ctc acc gtg cgg 930 Cys Leu Phe Gln Pro Leu Met Phe
Ile Asn Gly Ser Leu Thr Val Arg 270 275 280 gga gtt ccc gtg gag gcc
cgg cga ctg cgg gag cta aac cac att cgc 978 Gly Val Pro Val Glu Ala
Arg Arg Leu Arg Glu Leu Asn His Ile Arg 285 290 295 gag cac ctg aac
ctc ccg ctg gtg cga agt gcg gcg gcg gag gaa ccc 1026 Glu His Leu
Asn Leu Pro Leu Val Arg Ser Ala Ala Ala Glu Glu Pro 300 305 310 ggg
gcg ccc ctc acg acc ccg ccc gtc ctg cag ggc aac cag gcc cgc 1074
Gly Ala Pro Leu Thr Thr Pro Pro Val Leu Gln Gly Asn Gln Ala Arg 315
320 325 tcc tct ggg tac ttt atg ctg ctg atc cgg gcc aag ttg gac tcg
tac 1122 Ser Ser Gly Tyr Phe Met Leu Leu Ile Arg Ala Lys Leu Asp
Ser Tyr 330 335 340 345 tcc agc gtc gcg acc tcg gag ggc gag tcc gtc
atg cgg gag cac gcg 1170 Ser Ser Val Ala Thr Ser Glu Gly Glu Ser
Val Met Arg Glu His Ala 350 355 360 tat agc cgc ggg cgg acc aga aac
aat tac gga tcg aca atc gag ggc 1218 Tyr Ser Arg Gly Arg Thr Arg
Asn Asn Tyr Gly Ser Thr Ile Glu Gly 365 370 375 ctg ctc gac ctc ccg
gac gac gat gac gct cct gcg gag gcc ggg ctg 1266 Leu Leu Asp Leu
Pro Asp Asp Asp Asp Ala Pro Ala Glu Ala Gly Leu 380 385 390 gtg gcg
ccg cgc atg tcg ttt ctc tcc gcg gga caa cgc ccc cgc aga 1314 Val
Ala Pro Arg Met Ser Phe Leu Ser Ala Gly Gln Arg Pro Arg Arg 395 400
405 ctg tcc acc acc gcc ccc att acc gac gtc agc ctg gga gac gaa ctc
1362 Leu Ser Thr Thr Ala Pro Ile Thr Asp Val Ser Leu Gly Asp Glu
Leu 410 415 420 425 cgc ctg gac ggc gag gag gtg gat atg acg ccc gcc
gac gcc ctg gac 1410 Arg Leu Asp Gly Glu Glu Val Asp Met Thr Pro
Ala Asp Ala Leu Asp 430 435 440 gac ttc gac ttg gag atg ctg ggg gac
gtg gag tcc ccc tcc ccg gga 1458 Asp Phe Asp Leu Glu Met Leu Gly
Asp Val Glu Ser Pro Ser Pro Gly 445 450 455 atg acc cac gac ccc gtc
tcg tat ggg gct ttg gac gtg gac gat ttt 1506 Met Thr His Asp Pro
Val Ser Tyr Gly Ala Leu Asp Val Asp Asp Phe 460 465 470 gag ttt gaa
cag atg ttt acc gat gcc atg ggc att gac gac ttt ggg 1554 Glu Phe
Glu Gln Met Phe Thr Asp Ala Met Gly Ile Asp Asp Phe Gly 475 480 485
ggg tag gatgtgcgac cgggcggcgc gccccccccc caccaccgcc ccgcctcacc 1610
Gly * 490 tccgtctgta tcgcgataga gggttcgcaa ccacagcaat aaacattggc
aagcaactca 1670 tcatacgcgg cgtgcgttgg ctgtttatta cgggaccatg
aaagaaatgg ggttacgcgc 1730 ggggtggggg gtgtgtgccg ttgggttggg
cgttagtcgc gcctacgagc ccgcggtcgt 1790 gtagattcgc gtcacagaac
ggctcgtggt gctggggtcc gcgtataaag gcaggcgcgc 1850 gggtcccgtt
ctcgcatttg cccgcgggtc tgcgtgggga cgaggcccac ccccccaccc 1910
ttgttggagc ggtcgcgttt tctctgttcc cgtcgtgccg gttcctaccc cccgctccct
1970 gggaccgccc cctacccccc acctccccgt ttgggcctcc cccctcgcac
cacccctttc 2030 ctcgtccgtc tgcggggagg gcgtgtgtaa aaaatcgggc
ctccggccac catgtccgtg 2090 cgcgggcatg ccgtacgccg gaggcgcgcc
tccacccggt cccatgcccc gtccgcgcat 2150 cgcgccgact cgcccgtgga
ggacgagccc gagggcggtg gagtcgggtt aatggggtac 2210 c 2211 5 490 PRT
Herpes simplex virus type 2 5 Met Asp Leu Leu Val Asp Asp Leu Phe
Ala Asp Arg Asp Gly Val Ser 1 5 10 15 Pro Pro Pro Pro Arg Pro Ala
Gly Gly Pro Lys Asn Thr Pro Ala Ala 20 25 30 Pro Pro Leu Tyr Ala
Thr Gly Arg Leu Ser Gln Ala Gln Leu Met Pro 35 40 45 Ser Pro Pro
Met Pro Val Pro Pro Ala Ala Leu Phe Asn Arg Leu Leu 50 55 60 Asp
Asp Leu Gly Phe Ser Ala Gly Pro Ala Leu Cys Thr Met Leu Asp 65 70
75 80 Thr Trp Asn Glu Asp Leu Phe Ser Gly Phe Pro Thr Asn Ala Asp
Met 85 90 95 Tyr Arg Glu Cys Lys Phe Leu Ser Thr Leu Pro Ser Asp
Val Ile Asp 100 105 110 Trp Gly Asp Ala His Val Pro Glu Arg Ser Pro
Ile Asp Ile Arg Ala 115 120 125 His Gly Asp Val Ala Phe Pro Thr Leu
Pro Ala Thr Arg Asp Glu Leu 130 135 140 Pro Ser Tyr Tyr Glu Ala Met
Ala Gln Phe Phe Arg Gly Glu Leu Arg 145 150 155 160 Ala Arg Glu Glu
Ser Tyr Arg Thr Val Leu Ala Asn Phe Cys Ser Ala 165 170 175 Leu Tyr
Arg Tyr Leu Arg Ala Ser Val Arg Gln Leu His Arg Gln Ala 180 185 190
His Met Arg Gly Arg Asn Arg Asp Leu Arg Glu Met Leu Arg Thr Thr 195
200 205 Ile Ala Asp Arg Tyr Tyr Arg Glu Thr Ala Arg Leu Ala Arg Val
Leu 210 215 220 Phe Leu His Leu Tyr Leu Phe Leu Ser Arg Glu Ile Leu
Trp Ala Ala 225 230 235 240 Tyr Ala Glu Gln Met Met Arg Pro Asp Leu
Phe Asp Gly Leu Cys Cys 245 250 255 Asp Leu Glu Ser Trp Arg Gln Leu
Ala Cys Leu Phe Gln Pro Leu Met 260 265 270 Phe Ile Asn Gly Ser Leu
Thr Val Arg Gly Val Pro Val Glu Ala Arg 275 280
285 Arg Leu Arg Glu Leu Asn His Ile Arg Glu His Leu Asn Leu Pro Leu
290 295 300 Val Arg Ser Ala Ala Ala Glu Glu Pro Gly Ala Pro Leu Thr
Thr Pro 305 310 315 320 Pro Val Leu Gln Gly Asn Gln Ala Arg Ser Ser
Gly Tyr Phe Met Leu 325 330 335 Leu Ile Arg Ala Lys Leu Asp Ser Tyr
Ser Ser Val Ala Thr Ser Glu 340 345 350 Gly Glu Ser Val Met Arg Glu
His Ala Tyr Ser Arg Gly Arg Thr Arg 355 360 365 Asn Asn Tyr Gly Ser
Thr Ile Glu Gly Leu Leu Asp Leu Pro Asp Asp 370 375 380 Asp Asp Ala
Pro Ala Glu Ala Gly Leu Val Ala Pro Arg Met Ser Phe 385 390 395 400
Leu Ser Ala Gly Gln Arg Pro Arg Arg Leu Ser Thr Thr Ala Pro Ile 405
410 415 Thr Asp Val Ser Leu Gly Asp Glu Leu Arg Leu Asp Gly Glu Glu
Val 420 425 430 Asp Met Thr Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu
Glu Met Leu 435 440 445 Gly Asp Val Glu Ser Pro Ser Pro Gly Met Thr
His Asp Pro Val Ser 450 455 460 Tyr Gly Ala Leu Asp Val Asp Asp Phe
Glu Phe Glu Gln Met Phe Thr 465 470 475 480 Asp Ala Met Gly Ile Asp
Asp Phe Gly Gly 485 490 6 3694 DNA Saccharomyces cerevisiae CDS
(443)...(3088) GAL4 6 gatcccttaa gtttaaacaa caacagcaag caggtgtgca
agacactaga gactcctaac 60 atgatgtatg ccaataaaac acaagagata
aacaacattg catggaggcc ccagaggggc 120 gattggtttg ggtgcgtgag
cggcaagaag tttcaaaacg tccgcgtcct ttgagacagc 180 attcgcccag
tatttttttt attctacaaa ccttctataa tttcaaagta tttacataat 240
tctgtatcag tttaatcacc ataatatcgt tttctttgtt tagtgcaatt aatttttcct
300 attgttactt cgggcctttt tctgttttat gagctatttt ttccgtcatc
cttccccaga 360 ttttcagctt catctccaga ttgtgtctac gtaatgcacg
ccatcatttt aagagaggac 420 agagaagcaa gcctcctgaa ag atg aag cta ctg
tct tct atc gaa caa gca 472 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala
1 5 10 tgc gat att tgc cga ctt aaa aag ctc aag tgc tcc aaa gaa aaa
ccg 520 Cys Asp Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys
Pro 15 20 25 aag tgc gcc aag tgt ctg aag aac aac tgg gag tgt cgc
tac tct ccc 568 Lys Cys Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg
Tyr Ser Pro 30 35 40 aaa acc aaa agg tct ccg ctg act agg gca cat
ctg aca gaa gtg gaa 616 Lys Thr Lys Arg Ser Pro Leu Thr Arg Ala His
Leu Thr Glu Val Glu 45 50 55 tca agg cta gaa aga ctg gaa cag cta
ttt cta ctg att ttt cct cga 664 Ser Arg Leu Glu Arg Leu Glu Gln Leu
Phe Leu Leu Ile Phe Pro Arg 60 65 70 gaa gac ctt gac atg att ttg
aaa atg gat tct tta cag gat ata aaa 712 Glu Asp Leu Asp Met Ile Leu
Lys Met Asp Ser Leu Gln Asp Ile Lys 75 80 85 90 gca ttg tta aca gga
tta ttt gta caa gat aat gtg aat aaa gat gcc 760 Ala Leu Leu Thr Gly
Leu Phe Val Gln Asp Asn Val Asn Lys Asp Ala 95 100 105 gtc aca gat
aga ttg gct tca gtg gag act gat atg cct cta aca ttg 808 Val Thr Asp
Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu 110 115 120 aga
cag cat aga ata agt gcg aca tca tca tcg gaa gag agt agt aac 856 Arg
Gln His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn 125 130
135 aaa ggt caa aga cag ttg act gta tcg att gac tcg gca gct cat cat
904 Lys Gly Gln Arg Gln Leu Thr Val Ser Ile Asp Ser Ala Ala His His
140 145 150 gat aac tcc aca att ccg ttg gat ttt atg ccc agg gat gct
ctt cat 952 Asp Asn Ser Thr Ile Pro Leu Asp Phe Met Pro Arg Asp Ala
Leu His 155 160 165 170 gga ttt gat tgg tct gaa gag gat gac atg tcg
gat ggc ttg ccc ttc 1000 Gly Phe Asp Trp Ser Glu Glu Asp Asp Met
Ser Asp Gly Leu Pro Phe 175 180 185 ctg aaa acg gac ccc aac aat aat
ggg ttc ttt ggc gac ggt tct ctc 1048 Leu Lys Thr Asp Pro Asn Asn
Asn Gly Phe Phe Gly Asp Gly Ser Leu 190 195 200 tta tgt att ctt cga
tct att ggc ttt aaa ccg gaa aat tac acg aac 1096 Leu Cys Ile Leu
Arg Ser Ile Gly Phe Lys Pro Glu Asn Tyr Thr Asn 205 210 215 tct aac
gtt aac agg ctc ccg acc atg att acg gat aga tac acg ttg 1144 Ser
Asn Val Asn Arg Leu Pro Thr Met Ile Thr Asp Arg Tyr Thr Leu 220 225
230 gct tct aga tcc aca aca tcc cgt tta ctt caa agt tat ctc aat aat
1192 Ala Ser Arg Ser Thr Thr Ser Arg Leu Leu Gln Ser Tyr Leu Asn
Asn 235 240 245 250 ttt cac ccc tac tgc cct atc gtg cac tca ccg acg
cta atg atg ttg 1240 Phe His Pro Tyr Cys Pro Ile Val His Ser Pro
Thr Leu Met Met Leu 255 260 265 tat aat aac cag att gaa atc gcg tcg
aag gat caa tgg caa atc ctt 1288 Tyr Asn Asn Gln Ile Glu Ile Ala
Ser Lys Asp Gln Trp Gln Ile Leu 270 275 280 ttt aac tgc ata tta gcc
att gga gcc tgg tgt ata gag ggg gaa tct 1336 Phe Asn Cys Ile Leu
Ala Ile Gly Ala Trp Cys Ile Glu Gly Glu Ser 285 290 295 act gat ata
gat gtt ttt tac tat caa aat gct aaa tct cat ttg acg 1384 Thr Asp
Ile Asp Val Phe Tyr Tyr Gln Asn Ala Lys Ser His Leu Thr 300 305 310
agc aag gtc ttc gag tca ggt tcc ata att ttg gtg aca gcc cta cat
1432 Ser Lys Val Phe Glu Ser Gly Ser Ile Ile Leu Val Thr Ala Leu
His 315 320 325 330 ctt ctg tcg cga tat aca cag tgg agg cag aaa aca
aat act agc tat 1480 Leu Leu Ser Arg Tyr Thr Gln Trp Arg Gln Lys
Thr Asn Thr Ser Tyr 335 340 345 aat ttt cac agc ttt tcc ata aga atg
gcc ata tca ttg ggc ttg aat 1528 Asn Phe His Ser Phe Ser Ile Arg
Met Ala Ile Ser Leu Gly Leu Asn 350 355 360 agg gac ctc ccc tcg tcc
ttc agt gat agc agc att ctg gaa caa aga 1576 Arg Asp Leu Pro Ser
Ser Phe Ser Asp Ser Ser Ile Leu Glu Gln Arg 365 370 375 cgc cga att
tgg tgg tct gtc tac tct tgg gag atc caa ttg tcc ctg 1624 Arg Arg
Ile Trp Trp Ser Val Tyr Ser Trp Glu Ile Gln Leu Ser Leu 380 385 390
ctt tat ggt cga tcc atc cag ctt tct cag aat aca atc tcc ttc cct
1672 Leu Tyr Gly Arg Ser Ile Gln Leu Ser Gln Asn Thr Ile Ser Phe
Pro 395 400 405 410 tct tct gtc gac gat gtg cag cgt acc aca aca ggt
ccc acc ata tat 1720 Ser Ser Val Asp Asp Val Gln Arg Thr Thr Thr
Gly Pro Thr Ile Tyr 415 420 425 cat ggc atc att gaa aca gca agg ctc
tta caa gtt ttc aca aaa atc 1768 His Gly Ile Ile Glu Thr Ala Arg
Leu Leu Gln Val Phe Thr Lys Ile 430 435 440 tat gaa cta gac aaa aca
gta act gca gaa aaa agt cct ata tgt gca 1816 Tyr Glu Leu Asp Lys
Thr Val Thr Ala Glu Lys Ser Pro Ile Cys Ala 445 450 455 aaa aaa tgc
ttg atg att tgt aat gag att gag gag gtt tcg aga cag 1864 Lys Lys
Cys Leu Met Ile Cys Asn Glu Ile Glu Glu Val Ser Arg Gln 460 465 470
gca cca aag ttt tta caa atg gat att tcc acc acc gct cta acc aat
1912 Ala Pro Lys Phe Leu Gln Met Asp Ile Ser Thr Thr Ala Leu Thr
Asn 475 480 485 490 ttg ttg aag gaa cac cct tgg cta tcc ttt aca aga
ttc gaa ctg aag 1960 Leu Leu Lys Glu His Pro Trp Leu Ser Phe Thr
Arg Phe Glu Leu Lys 495 500 505 tgg aaa cag ttg tct ctt atc att tat
gta tta aga gat ttt ttc act 2008 Trp Lys Gln Leu Ser Leu Ile Ile
Tyr Val Leu Arg Asp Phe Phe Thr 510 515 520 aat ttt acc cag aaa aag
tca caa cta gaa cag gat caa aat gat cat 2056 Asn Phe Thr Gln Lys
Lys Ser Gln Leu Glu Gln Asp Gln Asn Asp His 525 530 535 caa agt tat
gaa gtt aaa cga tgc tcc atc atg tta agc gat gca gca 2104 Gln Ser
Tyr Glu Val Lys Arg Cys Ser Ile Met Leu Ser Asp Ala Ala 540 545 550
caa aga act gtt atg tct gta agt agc tat atg gac aat cat aat gtc
2152 Gln Arg Thr Val Met Ser Val Ser Ser Tyr Met Asp Asn His Asn
Val 555 560 565 570 acc cca tat ttt gcc tgg aat tgt tct tat tac ttg
ttc aat gca gtc 2200 Thr Pro Tyr Phe Ala Trp Asn Cys Ser Tyr Tyr
Leu Phe Asn Ala Val 575 580 585 cta gta ccc ata aag act cta ctc tca
aac tca aaa tcg aat gct gag 2248 Leu Val Pro Ile Lys Thr Leu Leu
Ser Asn Ser Lys Ser Asn Ala Glu 590 595 600 aat aac gag acc gca caa
tta tta caa caa att aac act gtt ctg atg 2296 Asn Asn Glu Thr Ala
Gln Leu Leu Gln Gln Ile Asn Thr Val Leu Met 605 610 615 cta tta aaa
aaa ctg gcc act ttt aaa atc cag act tgt gaa aaa tac 2344 Leu Leu
Lys Lys Leu Ala Thr Phe Lys Ile Gln Thr Cys Glu Lys Tyr 620 625 630
att caa gta ctg gaa gag gta tgt gcg ccg ttt ctg tta tca cag tgt
2392 Ile Gln Val Leu Glu Glu Val Cys Ala Pro Phe Leu Leu Ser Gln
Cys 635 640 645 650 gca atc cca tta ccg cat atc agt tat aac aat agt
aat ggt agc gcc 2440 Ala Ile Pro Leu Pro His Ile Ser Tyr Asn Asn
Ser Asn Gly Ser Ala 655 660 665 att aaa aat att gtc ggt tct gca act
atc gcc caa tac cct act ctt 2488 Ile Lys Asn Ile Val Gly Ser Ala
Thr Ile Ala Gln Tyr Pro Thr Leu 670 675 680 ccg gag gaa aat gtc aac
aat atc agt gtt aaa tat gtt tct cct ggc 2536 Pro Glu Glu Asn Val
Asn Asn Ile Ser Val Lys Tyr Val Ser Pro Gly 685 690 695 tca gta ggg
cct tca cct gtg cca ttg aaa tca gga gca agt ttc agt 2584 Ser Val
Gly Pro Ser Pro Val Pro Leu Lys Ser Gly Ala Ser Phe Ser 700 705 710
gat cta gtc aag ctg tta tct aac cgt cca ccc tct cgt aac tct cca
2632 Asp Leu Val Lys Leu Leu Ser Asn Arg Pro Pro Ser Arg Asn Ser
Pro 715 720 725 730 gtg aca ata cca aga agc aca cct tcg cat cgc tca
gtc acg cct ttt 2680 Val Thr Ile Pro Arg Ser Thr Pro Ser His Arg
Ser Val Thr Pro Phe 735 740 745 cta ggg caa cag caa cag ctg caa tca
tta gtg cca ctg acc ccg tct 2728 Leu Gly Gln Gln Gln Gln Leu Gln
Ser Leu Val Pro Leu Thr Pro Ser 750 755 760 gct ttg ttt ggt ggc gcc
aat ttt aat caa agt ggg aat att gct gat 2776 Ala Leu Phe Gly Gly
Ala Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp 765 770 775 agc tca ttg
tcc ttc act ttc act aac agt agc aac ggt ccg aac ctc 2824 Ser Ser
Leu Ser Phe Thr Phe Thr Asn Ser Ser Asn Gly Pro Asn Leu 780 785 790
ata aca act caa aca aat tct caa gcg ctt tca caa cca att gcc tcc
2872 Ile Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala
Ser 795 800 805 810 tct aac gtt cat gat aac ttc atg aat aat gaa atc
acg gct agt aaa 2920 Ser Asn Val His Asp Asn Phe Met Asn Asn Glu
Ile Thr Ala Ser Lys 815 820 825 att gat gat ggt aat aat tca aaa cca
ctg tca cct ggt tgg acg gac 2968 Ile Asp Asp Gly Asn Asn Ser Lys
Pro Leu Ser Pro Gly Trp Thr Asp 830 835 840 caa act gcg tat aac gcg
ttt gga atc act aca ggg atg ttt aat acc 3016 Gln Thr Ala Tyr Asn
Ala Phe Gly Ile Thr Thr Gly Met Phe Asn Thr 845 850 855 act aca atg
gat gat gta tat aac tat cta ttc gat gat gaa gat acc 3064 Thr Thr
Met Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr 860 865 870
cca cca aac cca aaa aaa gag taa aatgaatcgt agatactgaa aaaccccgca
3118 Pro Pro Asn Pro Lys Lys Glu * 875 880 agttcacttc aactgtgcat
cgtgcaccat ctcaatttct ttcatttata catcgttttg 3178 ccttctttta
tgtaactata ctcctctaag tttcaatctt ggccatgtaa cctctgatct 3238
atagaatttt ttaaatgact agaattaatg cccatctttt ttttggacct aaattcttca
3298 tgaaaatata ttacgagggc ttattcagaa gcttcgctca tataacgaaa
aaaaagggtt 3358 tggatcgaac gtaattgaga ttgattagtt aatactcaaa
ataaaacagc tcctaccacc 3418 agtgtaaagt agaacgttaa tagagcaatg
tcttcagaca aatctattga gaaaaataca 3478 gatacgatcg cctctgaagt
tcacgaaggt gataatcatt cgaataattt gggttcaatg 3538 gaggaagaga
taaaatcaac gccatcagac caatatgaag agatagctat aattccaact 3598
gagcccctcc attcggacaa agaactaaat gacaagcaac aaagtttagg ccatgaagca
3658 cccacaaatg tatcaagaga agaacctatt gggatc 3694 7 881 PRT
Saccharomyces cerevisiae 7 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala
Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys Ser Lys Glu
Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn Asn Trp Glu Cys
Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu Thr Arg Ala
His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 Glu Gln
Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80
Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85
90 95 Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu
Ala 100 105 110 Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His
Arg Ile Ser 115 120 125 Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys
Gly Gln Arg Gln Leu 130 135 140 Thr Val Ser Ile Asp Ser Ala Ala His
His Asp Asn Ser Thr Ile Pro 145 150 155 160 Leu Asp Phe Met Pro Arg
Asp Ala Leu His Gly Phe Asp Trp Ser Glu 165 170 175 Glu Asp Asp Met
Ser Asp Gly Leu Pro Phe Leu Lys Thr Asp Pro Asn 180 185 190 Asn Asn
Gly Phe Phe Gly Asp Gly Ser Leu Leu Cys Ile Leu Arg Ser 195 200 205
Ile Gly Phe Lys Pro Glu Asn Tyr Thr Asn Ser Asn Val Asn Arg Leu 210
215 220 Pro Thr Met Ile Thr Asp Arg Tyr Thr Leu Ala Ser Arg Ser Thr
Thr 225 230 235 240 Ser Arg Leu Leu Gln Ser Tyr Leu Asn Asn Phe His
Pro Tyr Cys Pro 245 250 255 Ile Val His Ser Pro Thr Leu Met Met Leu
Tyr Asn Asn Gln Ile Glu 260 265 270 Ile Ala Ser Lys Asp Gln Trp Gln
Ile Leu Phe Asn Cys Ile Leu Ala 275 280 285 Ile Gly Ala Trp Cys Ile
Glu Gly Glu Ser Thr Asp Ile Asp Val Phe 290 295 300 Tyr Tyr Gln Asn
Ala Lys Ser His Leu Thr Ser Lys Val Phe Glu Ser 305 310 315 320 Gly
Ser Ile Ile Leu Val Thr Ala Leu His Leu Leu Ser Arg Tyr Thr 325 330
335 Gln Trp Arg Gln Lys Thr Asn Thr Ser Tyr Asn Phe His Ser Phe Ser
340 345 350 Ile Arg Met Ala Ile Ser Leu Gly Leu Asn Arg Asp Leu Pro
Ser Ser 355 360 365 Phe Ser Asp Ser Ser Ile Leu Glu Gln Arg Arg Arg
Ile Trp Trp Ser 370 375 380 Val Tyr Ser Trp Glu Ile Gln Leu Ser Leu
Leu Tyr Gly Arg Ser Ile 385 390 395 400 Gln Leu Ser Gln Asn Thr Ile
Ser Phe Pro Ser Ser Val Asp Asp Val 405 410 415 Gln Arg Thr Thr Thr
Gly Pro Thr Ile Tyr His Gly Ile Ile Glu Thr 420 425 430 Ala Arg Leu
Leu Gln Val Phe Thr Lys Ile Tyr Glu Leu Asp Lys Thr 435 440 445 Val
Thr Ala Glu Lys Ser Pro Ile Cys Ala Lys Lys Cys Leu Met Ile 450 455
460 Cys Asn Glu Ile Glu Glu Val Ser Arg Gln Ala Pro Lys Phe Leu Gln
465 470 475 480 Met Asp Ile Ser Thr Thr Ala Leu Thr Asn Leu Leu Lys
Glu His Pro 485 490 495 Trp Leu Ser Phe Thr Arg Phe Glu Leu Lys Trp
Lys Gln Leu Ser Leu 500 505 510 Ile Ile Tyr Val Leu Arg Asp Phe Phe
Thr Asn Phe Thr Gln Lys Lys 515 520 525 Ser Gln Leu Glu Gln Asp Gln
Asn Asp His Gln Ser Tyr Glu Val Lys 530 535 540 Arg Cys Ser Ile Met
Leu Ser Asp Ala Ala Gln Arg Thr Val Met Ser 545 550 555 560 Val Ser
Ser Tyr Met Asp Asn His Asn Val Thr Pro Tyr Phe Ala Trp 565 570 575
Asn Cys Ser Tyr Tyr Leu Phe Asn Ala Val Leu Val Pro Ile Lys Thr 580
585 590 Leu Leu Ser Asn Ser Lys Ser Asn Ala Glu Asn Asn Glu Thr Ala
Gln 595 600 605 Leu Leu Gln Gln Ile Asn Thr Val Leu Met Leu Leu Lys
Lys Leu Ala 610 615 620 Thr Phe Lys Ile Gln Thr Cys Glu Lys Tyr Ile
Gln
Val Leu Glu Glu 625 630 635 640 Val Cys Ala Pro Phe Leu Leu Ser Gln
Cys Ala Ile Pro Leu Pro His 645 650 655 Ile Ser Tyr Asn Asn Ser Asn
Gly Ser Ala Ile Lys Asn Ile Val Gly 660 665 670 Ser Ala Thr Ile Ala
Gln Tyr Pro Thr Leu Pro Glu Glu Asn Val Asn 675 680 685 Asn Ile Ser
Val Lys Tyr Val Ser Pro Gly Ser Val Gly Pro Ser Pro 690 695 700 Val
Pro Leu Lys Ser Gly Ala Ser Phe Ser Asp Leu Val Lys Leu Leu 705 710
715 720 Ser Asn Arg Pro Pro Ser Arg Asn Ser Pro Val Thr Ile Pro Arg
Ser 725 730 735 Thr Pro Ser His Arg Ser Val Thr Pro Phe Leu Gly Gln
Gln Gln Gln 740 745 750 Leu Gln Ser Leu Val Pro Leu Thr Pro Ser Ala
Leu Phe Gly Gly Ala 755 760 765 Asn Phe Asn Gln Ser Gly Asn Ile Ala
Asp Ser Ser Leu Ser Phe Thr 770 775 780 Phe Thr Asn Ser Ser Asn Gly
Pro Asn Leu Ile Thr Thr Gln Thr Asn 785 790 795 800 Ser Gln Ala Leu
Ser Gln Pro Ile Ala Ser Ser Asn Val His Asp Asn 805 810 815 Phe Met
Asn Asn Glu Ile Thr Ala Ser Lys Ile Asp Asp Gly Asn Asn 820 825 830
Ser Lys Pro Leu Ser Pro Gly Trp Thr Asp Gln Thr Ala Tyr Asn Ala 835
840 845 Phe Gly Ile Thr Thr Gly Met Phe Asn Thr Thr Thr Met Asp Asp
Val 850 855 860 Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro Pro Asn
Pro Lys Lys 865 870 875 880 Glu 8 781 DNA Schistosoma bovis CDS
(6)...(641) GST 8 atacg atg act ggt gat cac atc aag gtt ata tat ttt
aac gga cgc gga 50 Met Thr Gly Asp His Ile Lys Val Ile Tyr Phe Asn
Gly Arg Gly 1 5 10 15 cga gct gaa tcg atc cgg atg aca ctt gtg gca
gct ggt gtg aac tac 98 Arg Ala Glu Ser Ile Arg Met Thr Leu Val Ala
Ala Gly Val Asn Tyr 20 25 30 gaa gat gag aga att agt ttc caa gat
tgg ccg aaa atc aaa cca act 146 Glu Asp Glu Arg Ile Ser Phe Gln Asp
Trp Pro Lys Ile Lys Pro Thr 35 40 45 att ccg ggc gga cga ttg cct
gca gtg aaa atc acc gat aat cat ggg 194 Ile Pro Gly Gly Arg Leu Pro
Ala Val Lys Ile Thr Asp Asn His Gly 50 55 60 cac gtg aaa tgg atg
tta gag agt ttg gct att gca cgg tat atg gcg 242 His Val Lys Trp Met
Leu Glu Ser Leu Ala Ile Ala Arg Tyr Met Ala 65 70 75 aag aag cat
cat atg atg gga gaa aca gac gag gag tat tat aat gtt 290 Lys Lys His
His Met Met Gly Glu Thr Asp Glu Glu Tyr Tyr Asn Val 80 85 90 95 gag
aag ttg att ggt cag gtt gaa gat cta gaa cat gaa tat cac aaa 338 Glu
Lys Leu Ile Gly Gln Val Glu Asp Leu Glu His Glu Tyr His Lys 100 105
110 act ttg atg aag cca gaa gaa gag aaa cag aag ata acc aaa gag ata
386 Thr Leu Met Lys Pro Glu Glu Glu Lys Gln Lys Ile Thr Lys Glu Ile
115 120 125 ctg aac ggc aaa gtg cca gtt ctt ctc gat att atc tgc gaa
tct ctg 434 Leu Asn Gly Lys Val Pro Val Leu Leu Asp Ile Ile Cys Glu
Ser Leu 130 135 140 aaa gcg tcc aca ggc aag ctg gct gtt ggg gat aaa
gtg act cta gcc 482 Lys Ala Ser Thr Gly Lys Leu Ala Val Gly Asp Lys
Val Thr Leu Ala 145 150 155 gac tta gtt ctg att gct gtc att gac cat
gtg act gat ctg gat aaa 530 Asp Leu Val Leu Ile Ala Val Ile Asp His
Val Thr Asp Leu Asp Lys 160 165 170 175 gaa ttt cta act ggc aag tat
cct gag atc cat aaa cat aga gaa aat 578 Glu Phe Leu Thr Gly Lys Tyr
Pro Glu Ile His Lys His Arg Glu Asn 180 185 190 cta tta gcc agt tca
ccg aga ttg gcg aaa tat tta tca gac agg gct 626 Leu Leu Ala Ser Ser
Pro Arg Leu Ala Lys Tyr Leu Ser Asp Arg Ala 195 200 205 gca act ccc
ttc tag aactgtcaac agaatgctgg gtgtgacgag attgaagata 681 Ala Thr Pro
Phe * 210 ttgatagtag tgcactggtg tgaccttttt acaaagacgt catttgtttt
atggtatttt 741 ttttcgcaat cgttattaaa ataaacttag ttttctgttt 781 9
211 PRT Schistosoma bovis 9 Met Thr Gly Asp His Ile Lys Val Ile Tyr
Phe Asn Gly Arg Gly Arg 1 5 10 15 Ala Glu Ser Ile Arg Met Thr Leu
Val Ala Ala Gly Val Asn Tyr Glu 20 25 30 Asp Glu Arg Ile Ser Phe
Gln Asp Trp Pro Lys Ile Lys Pro Thr Ile 35 40 45 Pro Gly Gly Arg
Leu Pro Ala Val Lys Ile Thr Asp Asn His Gly His 50 55 60 Val Lys
Trp Met Leu Glu Ser Leu Ala Ile Ala Arg Tyr Met Ala Lys 65 70 75 80
Lys His His Met Met Gly Glu Thr Asp Glu Glu Tyr Tyr Asn Val Glu 85
90 95 Lys Leu Ile Gly Gln Val Glu Asp Leu Glu His Glu Tyr His Lys
Thr 100 105 110 Leu Met Lys Pro Glu Glu Glu Lys Gln Lys Ile Thr Lys
Glu Ile Leu 115 120 125 Asn Gly Lys Val Pro Val Leu Leu Asp Ile Ile
Cys Glu Ser Leu Lys 130 135 140 Ala Ser Thr Gly Lys Leu Ala Val Gly
Asp Lys Val Thr Leu Ala Asp 145 150 155 160 Leu Val Leu Ile Ala Val
Ile Asp His Val Thr Asp Leu Asp Lys Glu 165 170 175 Phe Leu Thr Gly
Lys Tyr Pro Glu Ile His Lys His Arg Glu Asn Leu 180 185 190 Leu Ala
Ser Ser Pro Arg Leu Ala Lys Tyr Leu Ser Asp Arg Ala Ala 195 200 205
Thr Pro Phe 210 10 8 PRT Artificial Sequence 1X peptide 10 Asp Phe
Asp Leu Asp Met Leu Gly 1 5 11 16 PRT Artificial Sequence 2X
peptide 11 Asp Phe Asp Leu Asp Met Leu Gly Asp Phe Asp Leu Asp Met
Leu Gly 1 5 10 15 12 32 PRT Artificial Sequence 4X peptide 12 Asp
Phe Asp Leu Asp Met Leu Gly Asp Phe Asp Leu Asp Met Leu Gly 1 5 10
15 Asp Phe Asp Leu Asp Met Leu Gly Asp Phe Asp Leu Asp Met Leu Gly
20 25 30 13 227 PRT Artificial Sequence GST-2X 13 Met Thr Gly Asp
His Ile Lys Val Ile Tyr Phe Asn Gly Arg Gly Arg 1 5 10 15 Ala Glu
Ser Ile Arg Met Thr Leu Val Ala Ala Gly Val Asn Tyr Glu 20 25 30
Asp Glu Arg Ile Ser Phe Gln Asp Trp Pro Lys Ile Lys Pro Thr Ile 35
40 45 Pro Gly Gly Arg Leu Pro Ala Val Lys Ile Thr Asp Asn His Gly
His 50 55 60 Val Lys Trp Met Leu Glu Ser Leu Ala Ile Ala Arg Tyr
Met Ala Lys 65 70 75 80 Lys His His Met Met Gly Glu Thr Asp Glu Glu
Tyr Tyr Asn Val Glu 85 90 95 Lys Leu Ile Gly Gln Val Glu Asp Leu
Glu His Glu Tyr His Lys Thr 100 105 110 Leu Met Lys Pro Glu Glu Glu
Lys Gln Lys Ile Thr Lys Glu Ile Leu 115 120 125 Asn Gly Lys Val Pro
Val Leu Leu Asp Ile Ile Cys Glu Ser Leu Lys 130 135 140 Ala Ser Thr
Gly Lys Leu Ala Val Gly Asp Lys Val Thr Leu Ala Asp 145 150 155 160
Leu Val Leu Ile Ala Val Ile Asp His Val Thr Asp Leu Asp Lys Glu 165
170 175 Phe Leu Thr Gly Lys Tyr Pro Glu Ile His Lys His Arg Glu Asn
Leu 180 185 190 Leu Ala Ser Ser Pro Arg Leu Ala Lys Tyr Leu Ser Asp
Arg Ala Ala 195 200 205 Thr Pro Phe Asp Phe Asp Leu Asp Met Leu Gly
Asp Phe Asp Leu Asp 210 215 220 Met Leu Gly 225 14 243 PRT
Artificial Sequence GST-4X 14 Met Thr Gly Asp His Ile Lys Val Ile
Tyr Phe Asn Gly Arg Gly Arg 1 5 10 15 Ala Glu Ser Ile Arg Met Thr
Leu Val Ala Ala Gly Val Asn Tyr Glu 20 25 30 Asp Glu Arg Ile Ser
Phe Gln Asp Trp Pro Lys Ile Lys Pro Thr Ile 35 40 45 Pro Gly Gly
Arg Leu Pro Ala Val Lys Ile Thr Asp Asn His Gly His 50 55 60 Val
Lys Trp Met Leu Glu Ser Leu Ala Ile Ala Arg Tyr Met Ala Lys 65 70
75 80 Lys His His Met Met Gly Glu Thr Asp Glu Glu Tyr Tyr Asn Val
Glu 85 90 95 Lys Leu Ile Gly Gln Val Glu Asp Leu Glu His Glu Tyr
His Lys Thr 100 105 110 Leu Met Lys Pro Glu Glu Glu Lys Gln Lys Ile
Thr Lys Glu Ile Leu 115 120 125 Asn Gly Lys Val Pro Val Leu Leu Asp
Ile Ile Cys Glu Ser Leu Lys 130 135 140 Ala Ser Thr Gly Lys Leu Ala
Val Gly Asp Lys Val Thr Leu Ala Asp 145 150 155 160 Leu Val Leu Ile
Ala Val Ile Asp His Val Thr Asp Leu Asp Lys Glu 165 170 175 Phe Leu
Thr Gly Lys Tyr Pro Glu Ile His Lys His Arg Glu Asn Leu 180 185 190
Leu Ala Ser Ser Pro Arg Leu Ala Lys Tyr Leu Ser Asp Arg Ala Ala 195
200 205 Thr Pro Phe Asp Phe Asp Leu Asp Met Leu Gly Asp Phe Asp Leu
Asp 210 215 220 Met Leu Gly Asp Phe Asp Leu Asp Met Leu Gly Asp Phe
Asp Leu Asp 225 230 235 240 Met Leu Gly 15 345 PRT Artificial
Sequence His-tag-hTBP 15 His His His His His His Met Asp Gln Asn
Asn Ser Leu Pro Pro Tyr 1 5 10 15 Ala Gln Gly Leu Ala Ser Pro Gln
Gly Ala Met Thr Pro Gly Ile Pro 20 25 30 Ile Phe Ser Pro Met Met
Pro Tyr Gly Thr Gly Leu Thr Pro Gln Pro 35 40 45 Ile Gln Asn Thr
Asn Ser Leu Ser Ile Leu Glu Glu Gln Gln Arg Gln 50 55 60 Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 85
90 95 Gln Gln Gln Gln Gln Ala Val Ala Ala Ala Ala Val Gln Gln Ser
Thr 100 105 110 Ser Gln Gln Ala Thr Gln Gly Thr Ser Gly Gln Ala Pro
Gln Leu Phe 115 120 125 His Ser Gln Thr Leu Thr Thr Ala Pro Leu Pro
Gly Thr Thr Pro Leu 130 135 140 Tyr Pro Ser Pro Met Thr Pro Met Thr
Pro Ile Thr Pro Ala Thr Pro 145 150 155 160 Ala Ser Glu Ser Ser Gly
Ile Val Pro Gln Leu Gln Asn Ile Val Ser 165 170 175 Thr Val Asn Leu
Gly Cys Lys Leu Asp Leu Lys Thr Ile Ala Leu Arg 180 185 190 Ala Arg
Asn Ala Glu Tyr Asn Pro Lys Arg Phe Ala Ala Val Ile Met 195 200 205
Arg Ile Arg Glu Pro Arg Thr Thr Ala Leu Ile Phe Ser Ser Gly Lys 210
215 220 Met Val Cys Thr Gly Ala Lys Ser Glu Glu Gln Ser Arg Leu Ala
Ala 225 230 235 240 Arg Lys Tyr Ala Arg Val Val Gln Lys Leu Gly Phe
Pro Ala Lys Phe 245 250 255 Leu Asp Phe Lys Ile Gln Asn Met Val Gly
Ser Cys Asp Val Lys Phe 260 265 270 Pro Ile Arg Leu Glu Gly Leu Val
Leu Thr His Gln Gln Phe Ser Ser 275 280 285 Tyr Glu Pro Glu Leu Phe
Pro Gly Leu Ile Tyr Arg Met Ile Lys Pro 290 295 300 Arg Ile Val Leu
Leu Ile Phe Val Ser Gly Lys Val Val Leu Thr Gly 305 310 315 320 Ala
Lys Val Arg Ala Glu Ile Tyr Glu Ala Phe Glu Asn Ile Tyr Pro 325 330
335 Ile Leu Lys Gly Phe Arg Lys Thr Thr 340 345 16 233 PRT
Artificial Sequence His-tag-GST-2X 16 His His His His His His Met
Thr Gly Asp His Ile Lys Val Ile Tyr 1 5 10 15 Phe Asn Gly Arg Gly
Arg Ala Glu Ser Ile Arg Met Thr Leu Val Ala 20 25 30 Ala Gly Val
Asn Tyr Glu Asp Glu Arg Ile Ser Phe Gln Asp Trp Pro 35 40 45 Lys
Ile Lys Pro Thr Ile Pro Gly Gly Arg Leu Pro Ala Val Lys Ile 50 55
60 Thr Asp Asn His Gly His Val Lys Trp Met Leu Glu Ser Leu Ala Ile
65 70 75 80 Ala Arg Tyr Met Ala Lys Lys His His Met Met Gly Glu Thr
Asp Glu 85 90 95 Glu Tyr Tyr Asn Val Glu Lys Leu Ile Gly Gln Val
Glu Asp Leu Glu 100 105 110 His Glu Tyr His Lys Thr Leu Met Lys Pro
Glu Glu Glu Lys Gln Lys 115 120 125 Ile Thr Lys Glu Ile Leu Asn Gly
Lys Val Pro Val Leu Leu Asp Ile 130 135 140 Ile Cys Glu Ser Leu Lys
Ala Ser Thr Gly Lys Leu Ala Val Gly Asp 145 150 155 160 Lys Val Thr
Leu Ala Asp Leu Val Leu Ile Ala Val Ile Asp His Val 165 170 175 Thr
Asp Leu Asp Lys Glu Phe Leu Thr Gly Lys Tyr Pro Glu Ile His 180 185
190 Lys His Arg Glu Asn Leu Leu Ala Ser Ser Pro Arg Leu Ala Lys Tyr
195 200 205 Leu Ser Asp Arg Ala Ala Thr Pro Phe Asp Phe Asp Leu Asp
Met Leu 210 215 220 Gly Asp Phe Asp Leu Asp Met Leu Gly 225 230 17
249 PRT Artificial Sequence His-tag-GST-4X 17 His His His His His
His Met Thr Gly Asp His Ile Lys Val Ile Tyr 1 5 10 15 Phe Asn Gly
Arg Gly Arg Ala Glu Ser Ile Arg Met Thr Leu Val Ala 20 25 30 Ala
Gly Val Asn Tyr Glu Asp Glu Arg Ile Ser Phe Gln Asp Trp Pro 35 40
45 Lys Ile Lys Pro Thr Ile Pro Gly Gly Arg Leu Pro Ala Val Lys Ile
50 55 60 Thr Asp Asn His Gly His Val Lys Trp Met Leu Glu Ser Leu
Ala Ile 65 70 75 80 Ala Arg Tyr Met Ala Lys Lys His His Met Met Gly
Glu Thr Asp Glu 85 90 95 Glu Tyr Tyr Asn Val Glu Lys Leu Ile Gly
Gln Val Glu Asp Leu Glu 100 105 110 His Glu Tyr His Lys Thr Leu Met
Lys Pro Glu Glu Glu Lys Gln Lys 115 120 125 Ile Thr Lys Glu Ile Leu
Asn Gly Lys Val Pro Val Leu Leu Asp Ile 130 135 140 Ile Cys Glu Ser
Leu Lys Ala Ser Thr Gly Lys Leu Ala Val Gly Asp 145 150 155 160 Lys
Val Thr Leu Ala Asp Leu Val Leu Ile Ala Val Ile Asp His Val 165 170
175 Thr Asp Leu Asp Lys Glu Phe Leu Thr Gly Lys Tyr Pro Glu Ile His
180 185 190 Lys His Arg Glu Asn Leu Leu Ala Ser Ser Pro Arg Leu Ala
Lys Tyr 195 200 205 Leu Ser Asp Arg Ala Ala Thr Pro Phe Asp Phe Asp
Leu Asp Met Leu 210 215 220 Gly Asp Phe Asp Leu Asp Met Leu Gly Asp
Phe Asp Leu Asp Met Leu 225 230 235 240 Gly Asp Phe Asp Leu Asp Met
Leu Gly 245 18 15 PRT Artificial Sequence linker 18 Ser Ser Ser Ser
Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly 1 5 10 15 19 31 PRT
Artificial Sequence 1X-linker-1X 19 Asp Phe Asp Leu Asp Met Leu Gly
Ser Ser Ser Ser Gly Ser Ser Ser 1 5 10 15 Ser Gly Ser Ser Ser Ser
Gly Asp Phe Asp Leu Asp Met Leu Gly 20 25 30 20 231 PRT Artificial
Sequence His-tag-GAL4-VP16 20 His His His His His His Met Lys Leu
Leu Ser Ser Ile Glu Gln Ala 1 5 10 15 Cys Asp Ile Cys Arg Leu Lys
Lys Leu Lys Cys Ser Lys Glu Lys Pro 20 25 30 Lys Cys Ala Lys Cys
Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro 35 40 45 Lys Thr Lys
Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu 50 55 60 Ser
Arg Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg 65 70
75 80 Glu Asp Leu Asp Met Ile Leu Lys Met Asp Ser Leu Gln Asp Ile
Lys 85 90 95 Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn
Lys Asp Ala 100 105 110 Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp
Met Pro Leu Thr Leu 115 120 125 Arg Gln His Arg Ile Ser Ala Thr Ser
Ser Ser Glu Glu Ser Ser Asn 130 135 140 Lys Gly
Gln Arg Gln Leu Thr Val Ser Thr Ala Pro Ile Thr Asp Val 145 150 155
160 Ser Leu Gly Asp Glu Leu Arg Leu Asp Gly Glu Glu Val Asp Met Thr
165 170 175 Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Glu Met Leu Gly
Asp Val 180 185 190 Glu Ser Pro Ser Pro Gly Met Thr His Asp Pro Val
Ser Tyr Gly Ala 195 200 205 Leu Asp Val Asp Asp Phe Glu Phe Glu Gln
Met Phe Thr Asp Ala Met 210 215 220 Gly Ile Asp Asp Phe Gly Gly 225
230
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