Purification of tetracysteine-tagged proteins by affinity. Chromatography using a non-fluorescent, photochemically stable bisarsenical affinity ligand

Article abstract of DOI:10.1021/bc200038t

The use of genetically encoded small peptide tags such as polyhistidine and tetracysteine tags has become important for protein purification and enrichment. An improved affinity purification of tetracysteine (CCXXCC) tagged proteins has been achieved using a nonfluorescent, photochemically stable bisarsenical affinity ligand SplAsH. The photochemical stability of the SplAsHbiotin, shown in compound 5, is superior to FlAsH-EDT² and ReAsH-EDT². An application of the SplAsH tag for affinity purification of tetracysteine-tagged proteins is reported.

Full text of DOI:10.1021/bc200038t

TECHNICAL NOTE
pubs.acs.org/bc
Purification of Tetracysteine-Tagged Proteins by Affinity
Chromatography Using a Non-Fluorescent, Photochemically
Stable Bisarsenical Affinity Ligand
Lai-Qiang Ying* and Bruce P. Branchaud
Life Technologies, 29851 Willow Creek Road, Eugene, Oregon 97402, United States
ABSTRACT: The use of genetically encoded small peptide tags such as
polyhistidine and tetracysteine tags has become important for protein purification
and enrichment. An improved affinity purification of tetracysteine (CCXXCC)
tagged proteins has been achieved using a nonfluorescent, photochemically stable
bisarsenical affinity ligand SplAsH. The photochemical stability of the SplAsH-
biotin, shown in compound 5, is superior to FlAsH-EDT and ReAsH-EDT . An
2
2
application of the SplAsH tag for affinity purification of tetracysteine-tagged
proteins is reported.
1
5
’
INTRODUCTION
imidazole or low pH. This condition may disrupt some
16
protein structure and activity. In addition, the purified pro-
tein is often contaminated with intrinsic histidine-rich proteins
and small amounts of metal ions, which may inhibit many
Protein modification with reporter tags has allowed the study
of protein localization and proteinꢀprotein interactions inside
1
ꢀ3
living cells.
Traditionally, the protein of interest has to be
11,13
enzyme activities.
isolated in vitro, labeled with a reporter tag, then reintroduced
into cells by microinjection or electroporation. The use of
genetically encoded fluorescent proteins such as green fluores-
cent protein (GFP) has provided a simple and powerful tool to
study protein dynamics in cell biology. However, GFP is a large
protein (27 kDa), which could interfere with the physiological
The major limitation to using the FlAsH affinity matrix for
purification of tetracysteine-tagged proteins is a stability issue. The
compounds FlAsH-EDT and ReAsH-EDT are photochemically
2
2
17
4,5
unstable, which limits their wide application in purification of
tetracysteine-tagged proteins. We report herein on our successful
efforts to apply a photochemically stable bisarsenical affinity ligand
for affinity purification of tetracysteine-tagged proteins.
6,7
role of the protein of interest. Small peptide tags, such as the
hexahistidine (His ) tag and tetracysteine (CCPGCC) tag, have
6
greater advantage than GFP in protein labeling, because the small
6,8
tags have minimal interference with the protein of interest. The
tetracysteine tag combined with either fluorescein arsenical
’
EXPERIMENTAL PROCEDURES
hairpin binder (FlAsH-EDT , Figure 1) or Resorufin arsenical
2
General Materials and Methods. All chemicals and solvents
hairpin binder (ReAsH-EDT , Figure 1) provide useful tools for
2
were purchased from Aldrich, Acros, or TCI and were used
without further purification. Thin layer chromatography (TLC)
was performed on aluminum-backed plates with silica gel 60 with
F254 indicator. Flash column chromatography was carried out on
silica gel (230ꢀ400 mesh). High-performance liquid chromatog-
raphy (HPLC) analysis was performed on a Waters 600 instru-
ment equipped with a Waters photodiode array detector. All
moisture- or air-sensitive reactions were carried out under a static
9
ꢀ12
site-specific labeling of recombinant proteins inside live cells.
An especially important and useful feature of these molecules is
their fluorogenicity; the small molecules FlAsH-EDT2 and
ReAsH-EDT are membrane-permeant and almost nonfluores-
2
cent, so they can easily get into cells, then they bind with high
affinity and specificity to the tetracysteine domain, causing the
protein-bound dyes to become highly fluorescent.
1
On the basis of the high affinity and high specificity of the
interaction of biarsenical dyes with the tetracysteinemotif, methods
using biarsenicals immobilized on a matrix as a stationary phase for
affinity purification of tetracysteine-tagged proteins have been
argon atmosphere. H NMR (400 MHz) spectra were recorded at
room temperature on a Bruker ADVANCE-400 spectrometer
with residual proton resonances in deuterated solvents (DMSO-
d , or MeOD-d ) as the internal standard. LC/MS spectra were
6
4
1
1,13,14
developed.
The interaction between biarsenical dyes and
obtained with a Waters micromass ZQ instrument.
the tetracysteine motif can be easily reversed by incubation with
dithiols such as 1,2-ethanedithiol (EDT) or dithiothreitol (DTT).
This gives a gentle and mild elution condition with minimal
disruption of protein structure and activity.
0
0
0
tert-Butyl 2-(3 ,6 -dihydroxy-3-oxospiro[isoindoline-1,9 -
xanthene]-2-yl) ethylcarbamate (1). Fluorescein methyl
ester was prepared according to a literature procedure from
The most commonly used His -tag proteins are purified by
binding to metal ions (Ni or Co ) which are chelated to an
Received: January 19, 2011
6
2þ
2þ
Revised:
April 4, 2011
affinity matrix followed by elution with high concentrations of
Published: April 11, 2011
r 2011 American Chemical Society
987
dx.doi.org/10.1021/bc200038t
|Bioconjugate Chem. 2011, 22, 987–992

Bioconjugate Chemistry
TECHNICAL NOTE
yield) as a white solid. The obtained compound matches with data
17
in the literature.
0
0
0
0
2
-(2-Aminoethyl)-4 ,5 -di(1,3,2-dithiarsolan-2-yl)-3 ,6 -
0
dihydroxyspiro[isoindoline-1,9 -xanthen]-3-one (4). Com-
pound 3 (300 mg, 0.37 mmol) was dissolved in 8 mL CH Cl at
2
2
0
0
°C. TFA (4 mL) was added. The reaction mixture was stirred at
°C for 2 h. The reaction mixture was concentrated in vacuo. The
residue was precipitated in diethyl ether and dried under reduced
pressure via a vacuum pump to afford 4 (290 mg, 0.35 mmol, 95%
1
yield) as a yellow solid. H NMR (400 MHz, DMSO-d ): δ 10.01
6
(
s, 2H), 7.85 (dd, 1H, J = 2.0, 6.6 Hz), δ 7.60 (s, 2H), 7.56 (m,
2
H), 7.05 (dd, 1H, J = 2.0, 6.6 Hz), 6.52 (m, 4H), 3.50 (m, 8H),
.26 (t, 2H, J = 5.8 Hz), 2.39 (m, 2H). ESI-MS [MþH] m/z
þ
3
calcd. for 706.91, found 706.87.
N-(2-(4 ,5 -Di(1,3,2-dithiarsolan-2-yl)-3 ,6 -dihydroxy-3-
0
0
0
0
0
oxospiro[isoindoline-1,9 -xanthene]-2-yl)ethyl)-6-(5-((3aS,-
4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pen-
tanamido)hexanamide (5). Compound 4 (72 mg, 0.09 mmol)
and 6-((biotinoyl)amino)hexanoicacidsuccinimidylester(40 mg,
0.09 mmol, Life Technologies) were dissolved in 3 mL dry DMF.
TEA (37 μL, 0.26 mmol) was added. The reaction mixture was
stirred at room temperature for 1 h. Diethyl ether (20 mL) was
added to the reaction mixture. The precipitate was collected by
2 2
Figure 1. FlAsH-EDT , ReAsH-EDT , and related analogues.
18
fluorescein in 95% yield. Fluorescein methyl ester (1.0 g,
.9 mmol), and BocHNCH CH NH HCl (1.14 g, 5.8 mmol)
2
2
2
2
3
centrifuge and purified by column chromatography on SiO2
were dissolved in 10 mL DMF. Triethylamine (806 μL, 5.8 mmol)
was added. The reaction mixture was heated at 100 °C overnight in
a sealed pressure tube. After cooling to room temperature, the
solvent was evaporated in vacuo. The residue was dissolved in ethyl
(
CHCl /MeOH = 10:1) to afford 5 (80 mg, 0.08 mmol, 85%
3
1
yield) as a white solid. H NMR (400 MHz, DMSO-d ): δ 7.95
6
(
s, 2H), 7.90 (dd, 1H, J = 2.0, 6.6 Hz), 7.58 (m, 2H), 7.06 (dd, 1H,
J = 2.0, 6.6 Hz), 6.50 (m, 4H), 4.50 (m, 1H), 4.30 (m, 1H), 3.60
m, 8H), 3.18 (m, 5H), 2.99 (m, 3H), 2.71 (m, 1H), 2.19(t, J = 7.2
acetate (80 mL); washed with H O (40 mL), 0.1 M HCl (40 mL),
2
(
and brine (40 mL); dried over Na SO ; and concentrated in vacuo.
2
4
Hz, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.66ꢀ1.30 (m, 12H). ESI-MS
The crude mixture was purified by column chromatography on
þ
[
MþH] m/z calcd. for 1046.07, found 1046.34.
SiO (EtOAc/Hexane = 1:1ꢀ2:1) to afford 1 (1.12 g, 2.4 mmol,
2
Synthesis of Affinity Matrix 6. CrAsH-EDT succinimidyl
8
2% yield) as a white solid. The obtained compound matches with
2
1
1
17
ester was prepared according to a literature procedure. To a
20 mL plastic, fritted column with a Teflon stopcock and a cap to
seal the column, 10 mL of amino-agarose (Affi-Gel 102, Biorad)
in PBS buffer containing 0.1 mM EDTA, 5 mM β-mercaptoetha-
nol (BME), and 0.1 mM EDT, pH 7.4 was added, followed by
data that is in the literature.
tert-Butyl 2-(4 ,5 -bis(mercuric acetate)-3 ,6 -dihydroxy-3-
oxospiro[isoindoline-1,9 -xanthene]-2-yl) ethylcarbamate (2).
Compound 1 (1.1 g, 2.3 mmol) was dissolved in 60 mL ethanol,
0
0
0
0
0
then the solution was added to a solution of mercuric acetate
addition of 1 mL of CrAsH-EDT succinimidyl ester (10 mM
(
1.53 g, 4.8 mmol) in 4 mL HOAc and 100 mL H O. The
2
2
solution in DMSO, 10 μmol) and 0.5 mL of 1 M NaHCO . The
reaction mixture was stirred at 65 °C overnight in a sealed
pressure tube. After cooling to room temperature, the resulting
white precipitate was filtered through filter paper, washed
3
resin was mixed at RT for 4 h by end-to-end rotation, and washed
extensively with the same buffer. Capping of free amines was
achieved by treatment with a 10-fold excess of N-acetoxysucci-
nimide (prepared from acetic acid and N-hydroxysuccinimide
with H O (3 ꢁ 20 mL), and then dried under high vacuum
2
to afford 2 (2.05 g, 2.1 mmol, 90% yield) as a white solid.
The obtained compound matches with data that is in the
with dicyclohexylcarbodiimide in CH Cl ) over total amines
2 2
1
7
literature.
tert-Butyl 2-(4 ,5 -bis(1,3,2-dithiaarsolan-2-yl)-3 ,6 -dihy-
(∼12 μmol/mL) in the same phosphate buffer with slow
rotation for additional 2 h. The immobilized affinity matrix 6
was washed and stored in 50% EtOH at 4 °C.
0
0
0
0
0
droxy-3-oxospiro [isoindoline-1,9 -xanthene]-2-yl) ethylcar-
bamate (3). Compound 2 (1.0 g, 1.0 mmol) was suspended in
Synthesis of Affinity Matrix 7. To a 20 mL plastic, fritted
column with a Teflon stopcock and a cap to seal the column,
10 mL of N-hydroxysuccinimidyl ester (NHS) activated agarose
(Affi-Gel 10, Biorad) in 1/1 isopropanol/DMF was added,
followed by addition of 1 mL of compound 4 (20 mM solution
in DMSO, 20 μmol) and 0.2 mL of TEA. The resin was mixed at
RT for 2 h by end-to-end rotation, and washed with isopropanol
and DMF. Unreacted NHS groups were quenched by treatment
with 0.5 M ethanolamine in isopropanol for an additional 1 h.
The immobilized affinity matrix 7 was washed with PBS buffer
containing 0.1 mM EDTA, 5 mM BME, and 0.1 mM EDT, pH
7.4, and stored in 50% EtOH at 4 °C.
2
0mL THF. Di-isopropylethylamine (DIPEA, 1.4 mL, 8.0 mmol),
AsCl (1.6 mL, 20 mmol, caution: very toxic), and Pd(OAc) (20
3
2
mg) were added. The reaction mixture was stirred at 50 °C for 2 h
under argon, then at room temperature overnight under argon.
The reaction mixture was poured into acetone/aqueous phos-
phate buffer (1:1, 0.5 M K HPO , pH 7, 100 mL) containing
2
4
ethanedithiol (EDT, 3 mL). After stirring for 30 min, CHCl3
50 mL) was added, and the mixture was stirred for another 30
(
min. The organic layer was collected, and the aqueous layer was
washed with CHCl (3 ꢁ 30 mL). The combined organic layers
3
were dried over Na SO and concentrated in vacuo. The crude
2
4
mixture was purified by column chromatography on SiO (elute
Affinity Purification of Tetracysteine-Tagged Proteins
Using Affinity Matrixes 6 and 7. The GFP with a C-terminal
2
first with toluene to remove arsenicꢀEDT complex, then elute
with EtOAc/Hexane = 2:3) to afford 3 (760 mg, 0.94 mmol, 94%
CCPGCC tag as well as a N-terminal his tag was overexpressed
6
9
88
dx.doi.org/10.1021/bc200038t |Bioconjugate Chem. 2011, 22, 987–992

Bioconjugate Chemistry
TECHNICAL NOTE
a
Scheme 1. Synthesis of Nonfluorescent SplAsH-Biotin 5
a
Reaction conditions: (a) H
2
SO
4
, MeOH; BocHNCH
2
CH
2
2
NH HCl, TEA, DMF; (b) Hg(OAc)
2
, HOAc, EtOH/H
2
O; (c) AsCl
3
2
, DIPEA, Pd(OAc) ,
THF; ethandithiol, acetone/PBS; (d) TFA, DCM; (e) 6-((Biotinoyl)amino) hexanoic acid succinimidyl ester, TEA, DMF.
5
in Escherichia coli using standard methods. The collected cell
control, the SplAsH ligand coated tip was also incubated with a
buffer solution. The dissociation curve was generated by incubat-
ing these tips with the PBS buffer described above. The ForteBio
Data Analysis Octet software was used to process the curve by
subtracting a sample curve from the reference curve, and the
kinetic data were calculated by curve fitting.
pellet was lysed in 250 mM KCl, 50 mM KPO , 1 mM EDTA,
0 mM 2-mercaptoethane-sulfonate (MES), and 1 mM tris-
carboxyethyl) phosphine (TCEP), pH 7.5, by sonication. The
4
1
(
total protein concentration of the lysate was measured using
EZQ protein quantitation kit (Life Technologies), and GFP
concentration was quantified by GFP intrinsic fluorescence. The
freshly prepared GFP lysate (1 mL, ∼2.5 mg/mL) was added to
’
RESULTS AND DISCUSSION
0
.5 mL of affinity matrixes 6 and 7, respectively. The resin was
mixed at RT for 2 h by end-to-end rotation. The mixture was
transferred to a short column, the unbound material was
collected, and the resin was washed with buffer (250 mM KCl,
To improve the utility of carboxy-FlAsH-EDT (CrAsH-
2
EDT , Figure 1) in affinity purification of tetracysteine-tagged
2
11,20,21
proteins,
our focus is to apply a photochemically stable
5
3
0 mM KPO , 1 mM EDTA, 10 mM MES, 1 mM TCEP, pH 7.5,
FlAsHꢀEDT analogue. Recently, a fluoro-substituted FlAsH
4
2
ꢁ 2 mL), and then eluted with buffer (250 mM KCl, 50 mM
(F FlAsH, Figure 1) has been reported to dramatically improve
2
22
KPO , 1 mM EDTA, 10 mM 2,3-dimercapto-propanesulfonate
photostability. However, the synthesis of F FlAsH gives poor
4
2
(
(
DMPS), 3 ꢁ 0.5 mL). Fractions were analyzed by SDS-PAGE
overall yield (<4%), which limits its practical usefulness in
applications in affinity purification.
NuPAGE, 4ꢀ12% Bis-Tris Gel, Life Technologies).
Purification by the polyhistidine tag was done using the
We decided to focus on the use of a readily available
nonfluorescent SplAsH 4 (Spirolactam Arsenical Hairpin binder,
Scheme 1). SplAsH 4 was prepared according to the published
standard immobilized metal affinity chromatography method
(
(
incubated with 1 mL of Ni -NTA resin at RT for 1 h by end-to-
end rotation. The mixture was transferred to a short column, the
unbound material was collected, and the resin was washed with
buffer (50 mM NaH PO , 500 mM NaCl, 20 mM imidazole, pH
Life Technologies), as follows. The freshly prepared GFP lysate
17
1 mL) in 50 mM NaH PO , 500 mM NaCl, pH 8.0, was
procedure. Briefly, fluorescein was converted to fluorescein
2
4
2
þ
18
methyl ester in MeOH using H SO as a catalyst, then the ester
2
4
was reacted with mono Boc-protected ethylenediamine to give
23
fluorescein spirolactam 1. The fluorescein spirolactam 1 was
1
3
then converted to the bis-mercuric acetate derivative 2 and
then to the bis-arsenical derivative 3 by transmetalation with
2
4
8
.0, 3 ꢁ 5 mL) and then eluted with buffer (50 mM NaH PO ,
2
4
9
5
00 mM NaCl, 250 mM imidazole, pH 8.0, 3 ꢁ 0.5 mL).
palladium acetate as a catalyst. After cleavage of the Boc
Fractions were analyzed by SDS-PAGE (NuPAGE, 4ꢀ12%
protecting group with TFA, SplAsH 4 was obtained in 63%
overall yield in six steps. This synthesis can be easily scaled up for
the preparation of multigram amounts of 4. Compared to the
Bis-Tris Gel, Life Technologies).
Kinetic Measurement of Binding Affinity of SplAsH-biotin
11
5
and Tetracysteine-Tagged Protein (GFP). The kinetic mea-
synthesis of CrAsH-EDT (16% overall yield), the synthesis of
2
surement of binding affinity of SplAsH-biotin 5 and tetracys-
SplAsH 4 has great advantages in terms of easy preparation and
easy purification. The SplAsH 4 can be easily converted to
SplAsH-biotin 5. The absorbance maximum of SplAsH-biotin
5 was shifted from 509 to 242 nm with ε242 = 26 000 compared to
teine-tagged protein (GFP) was carried out using the ForteBio
19
system, which is a label-free, real-time detection system. The
streptavidin biosensor tips from ForteBio were prewet for 30 min
in water prior to use. All interaction analyses were conducted at
FlAsH-EDT (Figure 2), thus providing no inherent fluores-
2
2
5 °C in PBS buffer (pH 7.2) containing 5 mM MES, 0.1% BSA,
cence whether or not it is bound to the tetracysteine motif.
To test the photochemical stability, CrAsH-EDT2 and
SplAsH-biotin 5 were each prepared in 100 μM concentration
in degassed PBS buffer with 1 mM EDT, and 1 mL of each sample
in a sealed quartz curvette was irradiated with a xenon arc lamp at
and 0.05% Tween-20. The streptavidin biosensor tips were first
incubated with SplAsH-biotin 5 (50 μM) to immobilize the
SplAsH ligand onto the tips. Then, the SplAsH ligand coated tips
were incubated with purified tetracysteine-tagged GFP (50 nM)
in duplicate to generate the association curve. As a reference
2
250 mW/cm . Samples were checked by HPLC every 30 min,
9
89
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Bioconjugate Chemistry
TECHNICAL NOTE
Figure 4. Kinetics of association and dissociation of SplAsH-biotin 5
and tetracysteine-tagged GFP.
Figure 2. Absorbance spectra of FlAsH-EDT
biotin 5 (—) at PBS buffer.
2
(
) and SplAsH-
3
3 3
Figure 5. Affinity matrixes 6 and 7 used for purification of tetracysteine-
tagged proteins.
SplAsH 4 was conjugated with Affigel-10 (Biorad), and un-
reacted NHS groups were quenched with ethanolamine. We
chose the GFP with a C-terminal CCPGCC tag as well as a
N-terminal his tag as model test protein, because its recovery
6
yield can be easily quantified by measuring the intrinsic
fluorescence, and it can also be used to evaluate whether the
two different purification methods will affect the GFP fluores-
cence property. In separate, parallel experiments, the freshly
prepared GFP lysate was mixed with CrAsH-EDT affinity matrix 6
and the new nonfluorescent affinity matrix 7 (Figure 5) in a buffer
Figure 3. Photostability of CrAsH-EDT (O) and SplAsH-biotin 5 (b)
irradiated with a xenon arc lamp at 250 mW/cm .
2
2
5
,26
and the results are shown in Figure 3. The results clearly show
17
that the CrAsH-EDT exhibits poor photochemical stability.
containing 250 mM KCl, 50 mM KPO , 1 mM EDTA, 10 mM
2
4
SplAsH-biotin 5 exhibits remarkably improved photochemical
stability because blocking the quinoid tautomer formation by
spirolactam formation eliminates the excited state that serves as
MES, 1 mM TCEP, pH 7.5, for 2 h. The supernatant was collected
from each, then each resin was washed with buffer, followed by
eluting with buffer containing 10 mM DMPS. Fractions were
analyzed by SDS-PAGE. For another comparsion, the same protein
24
an intermediate in the photobleaching process.
2þ
In order to measure the binding affinity of SplAsH and
tetracysteine-tagged protein, SplAsH-biotin 5 was first immobi-
lized onto streptavidin sensor tips through biotinꢀstreptavidin
interaction. Then, the SplAsH coated sensor tips were incubated
with tetracysteine-tagged GFP to generate the association curve,
followed by incubating in buffer to get the dissociation curve
construct was purified by the conventional His :Ni -NTA
6
column.
It is well-known that the FlAsH ligand binds to the tetra-
cysteine motif with high affinity and specificity, and the binding
can be easily reversed by competing ligands, such as EDT,
1
1
DTT, and DMPS. These methods can be useful for the
(
Figure 4). On the basis of the on-and-off rate, the dissociation
purification of native tetracysteine-tagged proteins. However,
constant K was calculated to be approximately 2.4 ( 1.2 nM.
due to poor photochemical stability of CrAsH-EDT , the
D
2
Compared to the dissociation constant K (∼μM) for the
CrAsH-EDT affinity matrix 6 gave inconsistent results. In-
stead, SplAsH 4 displays superior photochemical stability. As
shown in Figure 6, by using the new nonfluorescent affinity
matrix 7 the desired protein was specifically captured and then
eluted with DMPS to afford highly purified protein (90%
purity) with reasonable yield (52% yield). Using the conven-
D
þ
25
interaction between His tag and Ni2 -NTA, the SplAsH ligand
has more than 1000-fold higher affinity (stronger, tighter
binding) and thus should be much more useful for the capture
and purification of low-abundance proteins.
To evaluate the application in affinity purification of tetra-
2
þ
cysteine-tagged proteins, the CrAsH-EDT succinimidyl ester
tional His :Ni -NTA method, the GFP was isolated in 92%
2
6
was coupled to Affigel-102 (Biorad), and the remaining free
amines were capped with excess of N-acetoxysuccinimide.
purity and 70% recovery yield. The lower recovery yield is
probably due to a stability issue of tetracysteine tag in vitro, by
1
1
9
90
dx.doi.org/10.1021/bc200038t |Bioconjugate Chem. 2011, 22, 987–992

Bioconjugate Chemistry
TECHNICAL NOTE
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(
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(
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6
Figure 6. Affinity purification of a tetracysteine-tagged and his -
2
þ
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(
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stained with SYPRO-Ruby.
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(
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’
CONCLUSION
(
(
In summary, the superior photochemical stability of SplAsH 4
compared to CrAsH-EDT can be applied for a new, improved
2
(
method for protein affinity purification. SplAsH 4 can be easily
prepared in large scale and high yield. SplAsH 4 retains high
affinity and specificity for tetracysteine tags. This provides better
affinity purification of tetracysteine-tagged proteins with SplAsH
non-denaturing isolation of an RNA polymerase enzyme complex
using an improved multi-use affinity probe resin. Mol. Biosyst. 1,
5
3–56.
(15) Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975)Metal
4
than with CrAsH-EDT . In addition, SplAsH 4 is nonfluor-
chelate affinity chromatography, a new approach to protein fractiona-
2
escent whether or not it is bound to tetracysteine tags. This
quenched property of SplAsH 4 provides an opportunity for
potential surface-based applications by immobilizing SplAsH 4
onto beads or array surfaces to capture tetracysteine-tagged
proteins, then performing protein detection, quantitation, and
functional assays using dye-labeled detection antibody. Detailed
studies of surface-based applications are currently underway.
tion. Nature 258, 598–599.
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small fluorescent molecules based on metal-chelation methodology.
Sensors 8, 1004–1024.
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tagged proteins with a SplAsH of color: a modular approach to bis-
arsenical fluorophores. ChemBioChem 8, 1642–1645.
(
18) Adamczyk, M., Grote, J., and Moore, J. A. (1999)Chemoenzy-
0
matic synthesis of 3 -O-(carboxyalkyl)fluorescein labels. Bioconjugate
Chem. 10, 544–547.
’
AUTHOR INFORMATION
(
19) Abdiche, Y., Malashock, D., Pinkerton, A., and Pons, J. (2008)
Corresponding Author
Determining kinetics and affinities of protein interactions using a
parallel real-time label-free biosensor, the Octet. Anal. Biochem.
77, 209–217.
*
E-mail: lai-qiang.ying@lifetech.com. Telephone: (541) 335-
158. FAX: (541) 335-0206.
0
3
(20) Cao, H., Chen, B., Squier, T. C., and Mayer, M. U. (2006)
CrAsH: a biarsenical multi-use affinity probe with low non-specific
fluorescence. Chem. Commun. 2601–2603.
’
ACKNOWLEDGMENT
We thank Michael O’Grady for assistance with GFP protein
expression.
(21) Adams, S. R., and Tsien, R. Y. (2008)Preparation of the
membrane-permeant biarsenicals FlAsH-EDT and ReAsH-EDT for
2
2
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527–1534.
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