ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 347 (2005) 129–134
www.elsevier.com/locate/yabio
A live cell hormone-binding assay on transgenic bacteria
expressing a eukaryotic receptor protein
Georgy A. Romanov a,b,¤, Lukán Spíchal c, Sergey N. Lomin b, Miroslav Strnad c,
Thomas Schmülling a
a
Free University of Berlin, Institute of Biology/Applied Genetics, D-14195 Berlin, Germany
b
Institute of Plant Physiology, Russian Academy of Sciences, 127276 Moscow, Russia
c
Institute of Experimental Botany, CAS, and Laboratory of Growth Regulators, Palacký University, CZ-78371 Olomouc, Czech Republic
Received 19 August 2005
Available online 30 September 2005
Abstract
The investigation of hormone–receptor interaction normally needs isolation and extensive puriWcation of the receptor protein or a
particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we have established a live cell-based
assay using transgenic bacteria expressing single eukaryotic receptors. Here we describe some biochemical features of the Arabidopsis
cytokinin receptor CRE1/AHK4 expressed in Escherichia coli. The data show that the main characteristics of the ligand–receptor interaction, including binding aYnity and ligand speciWcity, can be determined using intact bacteria expressing a functional receptor.
 2005 Elsevier Inc. All rights reserved.
Keywords: Hormone; Receptor; Cytokinin; Binding assay; Transgenic Escherichia coli
The hormone–receptor interaction is a key stage for
hormone signaling in the cell; therefore, the characteristics of such an interaction (binding) are of great importance. The binding data serve as ultimate proof of the
receptor function of a presumable sensor protein. The
hormone–receptor interaction is usually studied using
labeled hormone and isolated receptor protein or a particular fraction containing this receptor [1]. In most cases,
the procedure of receptor or fraction isolation and puriWcation is time-consuming, needs expensive equipment and
chemicals, and leads to unstable receptor preparations.
The subsequent binding assay with puriWed receptor or
fraction also needs additional equipment to discriminate
between bound and unbound hormones. These diYculties
become even greater in studies of membrane receptors,
which often lose their stability and hormone-binding
*
Corresponding author. Fax: +7 095 9778018.
E-mail address: [email protected] (G.A. Romanov).
0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.09.012
capacity on puriWcation [2]. In this article, we describe a
way in which to accomplish binding assays with a particular membranous receptor without its isolation. The proposed method is based on the use of intact transgenic
bacteria (Escherichia coli) expressing a functionally active
eukaryotic receptor protein, for example, the membranelocated sensor histidine kinase CRE1/AHK4 that senses
the phytohormone cytokinin in Arabidopsis thaliana [3,4].
Cytokinin binds to the predicted approximately 270amino acid extracellular region of the receptor, the socalled CHASE domain [5]. We took advantage of the fact
that this receptor was shown to enable E. coli to sense
cytokinin [4,6,7] and must, therefore, be present in a functional form. Here we show examples of the use of living
transgenic bacteria to characterize the interaction of the
receptor with cognate ligands and compare the binding
data with results obtained from isolated membranes. The
results show that this rapid and inexpensive approach
allows the determination of the most important characteristics of the hormone–receptor interaction.
130
Live cell hormone-binding assay / G.A. Romanov et al. / Anal. Biochem. 347 (2005) 129–134
Materials and methods
Bacterial strains and growth conditions
The E. coli strain KMI001 carrying the vector
pINIIIEH, which expresses the Arabidopsis histidine
kinase CRE1/AHK4 [4,6], was used in the experiments.
This vector provides the bacterial promoter and ribosomebinding sites for expressing any heterologous gene. The
E. coli strain KMI001 carrying the empty vector pINIII
was used as a control. Bacterial strains were kindly provided by T. Mizuno (Nagoya, Japan). Bacteria were grown
overnight in M9 medium (pH 7) with 0.1% casamino acids
[8] and 25 g/ml carbenicillin at 24 § 1 °C.
Live cell hormone-binding assay
For the binding assay, a homogeneous bacterial suspension was aliquoted (0.75 ml) to Eppendorf tubes containing
2.5 l of tritium-labeled trans-zeatin with or without additional unlabeled zeatin. Highly labeled trans-[2-3H]zeatin
(592 GBq/mmol) was obtained from the Isotope Laboratory of the Institute of Experimental Botany (Prague,
Czech Republic). Radiochemical purity was greater than
99%. For one probe, 1–2 pmol of trans-[2-3H]zeatin
(»20,000–40,000 cpm) was used. Routine incubations were
done in an ice bath for 30 min. After incubation, bacteria
were pelleted at 13.000 rpm for 2 min in the cold, supernatants were carefully vacuum aspirated, and 0.24 ml of 99%
ethanol was added to each pellet. The pellet was extracted
for at least 1 h with shaking and brieXy centrifuged, and the
radioactivity of 0.2 ml of ethanol extract was determined in
ACS-II scintillation cocktail (Amersham Biosciences, UK).
The analysis of the supernatant [9] showed the absence of
protein after bacteria centrifugation.
Microsome isolation
Membrane isolation from the E. coli strain expressing
CRE1/AHK4 was carried out as follows. All steps were
done at 4 °C, and all solutions used were precooled. One
liter of bacterial culture grown at 25 °C to OD600 » 1 was
pelleted, and the pellet was resuspended in 200 ml of cold
0.1 M Tris–HCl (pH 6.8) supplemented with 18% sucrose
and pelleted again. The pellet was again resuspended in the
same buVer, and the sucrose content was decreased to 10%.
Then 100 ml of cold 4 mM ethylenediamine tetraacetic acid
(EDTA,1 pH 7.5) containing lysozyme (0.2 mg/ml) was
added, and the suspension was incubated 15 min while
swirling. Then 2 ml of 1 M MgCl2 was added, and centrifugation at 10,000g for 20 min was used to separate periplasm
(supernatant) and spheroplasts (pellet). The periplasm was
stored at 4 °C, and spheroplasts were further processed by
1
Abbreviations used: EDTA, ethylenediamine tetraacetic acid; PMSF,
phenylmethanesulfonyl Xuoride; BSA, bovine serum albumin.
resuspending in 19 ml of 100 mM Tris–HCl (pH 7.8) supplemented with 18% sucrose and 2 mM EDTA. The volume
was Wlled up to 20 ml with DNase (10 g/ml), RNase (10 g/
ml), MgCl2 (10 M), phenylmethanesulfonyl Xuoride
(PMSF, 1 mM), leupeptin (10 g/ml) and aprotinin (10 g/
ml). Then the cells were lysed by sonication. The lysate was
pelleted at 3500g for 15 min, and the supernatant was further centrifuged at 50,000g for 1 h to separate cytoplasm
(supernatant) and membranes (pellet). Pellet was resuspended in 10 ml of 10 mM Tris–HCl (pH 6.8) containing
0.25 M sucrose, 2 mM EDTA, and 1 mM MgCl2 and was
pelleted again at 50,000g for 2 h. Cytoplasm (supernatant)
was removed, and the membranes were pooled in 10 ml of
10 mM Hepes (pH 7.4). All fractions were directly assayed
in binding assay or stored at 4 °C. The protein content in
fractions was determined according to Ref. [9] with bovine
serum albumin (BSA) as the standard.
Binding assay on microsomes
For the binding assay, 200 g of membrane proteins was
diluted in 10 mM Hepes (pH 7.4) containing BSA (1 mg/ml)
and dialyzed against the same buVer supplemented with
2 nM [2-3H]zeatin in the presence or absence of increasing
concentrations of the studied compound in a Dianorm
equilibrium dialyzer (Dianorm–Geräte, Munich, Germany)
for 20 h at 4 °C. Radioactivity of the dialysates was measured by scintillation counting using the Beckman LS 6500
scintillation counter (Beckman, Ramsey, MN, USA).
Statistics
Each data point of experiments represents the average of
two or three independent determinations. The signiWcance
of diVerences between mean values was evaluated using a
t test. Experiments were performed at least in duplicate
with similar results.
Results and discussion
One of the most important tasks in a study of hormone–
receptor complex formation is the quantitative discrimination between bound and unbound hormones. Bound hormone should, in turn, be assigned to speciWc and/or
nonspeciWc binding [1]. The commonly used approach is to
make the receptor insoluble while the free hormone
remains fully soluble. Soluble and insoluble phases are easily separated, such as by appropriate centrifugation, allowing the hormone fraction bound to receptors and the
unbound fraction to be discriminated quantitatively. When
a given receptor is expressed inside bacteria, it remains in
the insoluble phase, whereas unbound hormone is always
located in the soluble phase except for a minor part that
can be mechanically trapped by bacterial cells—the socalled nonspeciWc binding. Receptors that are expressed in
bacteria are easily accessible to hormones, and the bacterial
suspension can be aliquoted quantitatively to produce stan-
Live cell hormone-binding assay / G.A. Romanov et al. / Anal. Biochem. 347 (2005) 129–134
Fig. 1. Kinetics of [3H]zeatin binding to the E. coli CRE1/AHK4-expressing clone at diVerent temperatures. TB, total binding; NS, nonspeciWc
binding in the presence of approximately 5000-fold excess of unlabeled
trans-zeatin.
800
Bond [ 3 H]trans-zeatin (cpm)
dard probes. These properties of receptor-synthesizing bacteria provide all of the necessary prerequisites for a simple
binding assay.
In our experiments, we used transgenic E. coli expressing
the Arabidopsis cytokinin receptor CRE1/AHK4 [4,5]. The
functional activity of the receptor was proven previously in
cytokinin-dependent promoter activation tests because the
CRE1/AHK4 histidine kinase couples to an E. coli signaling system with a cps::lacZ fusion gene as target [4,6]. An
E. coli strain carrying only the empty vector was used as a
control. The presence of cytokinin-binding activity in
microsomes of CRE1/AHK4-expressing clones, but not in
control clones, was demonstrated in preliminary experiments. Bacteria were grown overnight in liquid medium up
to an OD600 close to 1. Then aliquots of the bacterial suspension were incubated with tritium-labeled hormone,
trans-[2-3H]zeatin. To discriminate between speciWc saturable binding and nonspeciWc nonsaturable binding, each
tube with labeled [3H]zeatin had its counterpart where
[3H]zeatin was mixed with a large (»5000-fold) excess of
nonlabeled trans-zeatin. Tubes with only [3H]zeatin were to
measure total binding (TB), whereas tubes with an excess of
nonlabeled zeatin were to determine nonspeciWc binding
(NS). The reliable diVerence between total binding and
nonspeciWc binding would indicate the presence of speciWc
binding (SB) sites [1].
For the Wrst assays, 0.75-ml aliquots of bacterial suspension were mixed with hormones and incubated for
30 min with periodic shaking. Then bacteria were pelleted
by brief centrifugation and supernatants were carefully
removed by vacuum aspiration. The pellets were used
directly for radioactivity determination without additional washing.
The results showed large diVerences between total binding and nonspeciWc binding in the case of the CRE1/AHK4expressing clone but not in the case of the control clone
(Table 1). This result was obtained in several repetitions
and variable assay conditions. This indicated that the cyto-
131
TB
600
400
SB
NS
200
0
6,0
6,5
7,0
7,5
8,0
pH
Table 1
Binding of [3H] trans-zeatin to the E. coli strain KMI001, expressing the
CRE1/AHK4 receptor gene (pINIII–AHK4) or harboring the empty vector (pINIII)
Total
binding
NonspeciWc
bindingb
SpeciWc
bindingc
Vector
Experiment
numbera
PINIII–AHK4
1
2
3
665.0 § 6
733.5 § 41
773.5 § 38
98.0 § 8
106.5 § 7
116.5 § 15
567.0
627.0
657.0
pINIII
1
2
3
89.0 § 7
98.5 § 9
161.5 § 24
85.0 § 2
99.5 § 21
179.0 § 2
2.0
¡1.0
¡17.5
a
Results of several independent experiments are shown. Data are cpm
of radiolabeled zeatin.
b
NonspeciWc binding was determined in the presence of approximately
5000-fold excess of unlabeled trans-zeatin.
c
The resulting speciWc binding represents the diVerence between total
binding and nonspeciWc binding.
Fig. 2. pH dependence of [3H]zeatin binding to the E. coli CRE1/AHK4expressing clone. The pH values were adjusted by adding small volumes of
concentrated NaOH or HCl to the bacterial suspension. SB, speciWc binding. For other abbreviations, see Fig. 1.
kinin-speciWc binding in CRE1/AHK4 clones was due to the
expression of the eukaryotic cytokinin receptor. Thereafter,
we studied diVerent parameters in more detail.
Fig. 1 shows the [3H]zeatin-binding kinetics at diVerent
temperatures (0, 20, and 37 °C). The total binding was inXuenced by the incubation temperature, which had a marginal
inXuence on nonspeciWc binding. The speciWc binding proceeded rapidly and reached an apparent equilibrium after
5–15 min (more rapidly at higher temperatures). At 37 °C, a
longer incubation time (60 min) led to some decrease
in binding (Fig. 1). On the whole, the level of bound hormone seemed to be rather stable during the entire
132
Live cell hormone-binding assay / G.A. Romanov et al. / Anal. Biochem. 347 (2005) 129–134
2000
1500
1000
3
Bond [ H]trans-zeatin (cpm)
period (5–60 min) of the assay. The highest level of speciWcally bound zeatin was observed at 0 °C (ice bath); therefore, all subsequent binding assays were performed at this
temperature.
The pH dependence of binding is shown in Fig. 2. The
nonspeciWc binding showed a tendency to rise at pH
extremes, but no drastic diVerences in speciWc binding was
observed in the pH range between 6 and 8. The binding at
pH 7 was close to the optimum; therefore, we continued
binding experiments at the pH of the standard M9 medium
(i.e., pH 7).
The competition of various signaling molecules with
[3H]zeatin for binding sites on the bacteria (Fig. 3) revealed
a high degree of speciWcity; among various compounds,
including auxins, abscisic acid, gibberellic acid, and some
others, only cytokinin was able to diminish the bound
radioactivity. The binding of various concentrations of
[3H]zeatin to the CRE1/AHK4-expressing bacteria is shown
in Fig. 4. The result clearly shows that the binding is saturable, indicating that the number of binding sites is limited. A
Scatchard analysis of these data yielded an apparent aYnity constant (Kd) of approximately 2.5 nM for CRE1/
AHK4 to zeatin (Fig. 4), and this value was conWrmed in
several independent experiments. This value is close to that
reported earlier (»4.5 nM) for 3H-labeled isopentenyl adenine interaction with isolated Schizosaccharomyces pombe
membranes containing the CRE1/AHK4 receptor [6]. We
have also determined the Kd of the zeatin–CRE1/AHK4
interaction using partially puriWed microsomes from
the CRE1/AHK4-expressing bacterial clone. The Kd values
obtained on microsomes (»6 nM) were close to
those obtained with bacteria, conWrming the validity of
results obtained with intact bacterial cells.
The concentration of cytokinin-binding sites in the suspension of transgenic bacteria was close to 0.1–0.2 nM. This
value corresponds to 0.5 to 1.2 £ 1014 sites in 0.75 ml of the
bacterial suspension used in the standard assay. If one
500
0
tZ
IAA
2,4-D
ABA
GA
cAMP
Spe
Control
Fig. 3. Competition of various signaling substances with [3H]zeatin for the
binding to CRE1/AHK4-expressing bacteria. All substances were tested in
the same concentration (8.6 M) except spermine (17.3 M). tZ, trans-zeatin; IAA and 2,4-D, auxins; ABA, abscisic acid; GA, gibberellic acid;
cAMP, cyclic AMP; Spe, spermine.
Fig. 4. Binding of various amounts of [3H]zeatin to CRE1/AHK4-expressing bacteria. Original data and Scatchard plot (inset) are shown. For
abbreviations, see Figs. 1 and 2 legends.
assumes that each receptor possesses one ligand-binding
site [5], then the number of CRE1/AHK4 receptors per
transgenic bacterial cell is calculated to be approximately
50,000–120,000. The speciWcally bound radioactivity was
approximately 1.5 £ 10¡4 Bq per bacterium.
The ligand speciWcity of CRE1/AHK4 was tested by
comparing the abilities of various cytokinin analogues to
compete with [3H]zeatin for CRE1/AHK4 binding. Five
diVerent zeatin-related compounds—trans-zeatin, cis-zeatin, zeatin riboside, dihydrozeatin, and zeatin O-glucoside—were tested in a concentration range of 0.025–2.5 M.
The resulting displacement curves (Fig. 5A) indicate that
trans-zeatin has the highest aYnity for the receptor. Zeatin
riboside, dihydrozeatin, and cis-zeatin display decreasing
aYnities, and zeatin O-glucoside was not able to displace
[3H]zeatin. When using puriWed CRE1/AHK4-containing
microsomes, the same order of hormonal activity of
compounds was obvious: trans-zeatin > zeatin riboside >
dihydrozeatin > cis-zeatin > zeatin O-glucoside (Fig. 5B).
The same activity range of these zeatin derivatives was
obtained previously using a reporter gene assay on the
CRE1/AHK4-expressing E. coli clone [7], conWrming the
validity of binding data. These results are also in good
agreement with the relative physiological activities of these
zeatin-type cytokinins [10].
The study has shown that this simple and quick binding
assay using intact transgenic bacteria expressing a eukaryotic receptor protein provides a convenient and reliable tool
for the investigation of hormone–receptor interaction. The
most important experiments that are commonly performed
using puriWed receptor preparations (e.g., determination of
binding aYnity, ligand speciWcity, inXuence of environment)
are fully feasible with receptors integrated into a bacterial
membrane. In some cases (including ours), the functional
Live cell hormone-binding assay / G.A. Romanov et al. / Anal. Biochem. 347 (2005) 129–134
133
Fig. 5. Competition of various cytokinin compounds with [3H]zeatin binding to the CRE1/AHK4 receptor in a bacterial suspension (A) and on partially
puriWed microsomes (B). tZ, trans-zeatin; cZ, cis-zeatin; tZR, trans-zeatin riboside; DZ, dihydrozeatin; ZOG, zeatin O-glucoside. For other abbreviations,
see Fig. 1 legend.
activity of integrated receptor can be proven by some kind
of reporter gene assay. In this respect, the intact living system oVers an advantage over traditional assays using puriWed receptor preparations. This holds true especially for
membranous (plasma membrane) receptors that often have
reduced stability on isolation and puriWcation. In bacterial
cells, such receptors not only remain stable and functionally
active but also are easily accessible for hormones due to
localization in the plasma membrane. The common problem
of contamination by other binding moieties, such as by
other receptors for the same hormone, has no importance in
a bacterial assay where only a deWned eukaryotic protein is
expressed. Another problem of binding assays might result
from hormone and/or receptor degradation and modiWcation during the binding procedure. In our case, the use of
bacteria (E. coli) that are evolutionary very far from higher
organisms and do not synthesize or use their hormones
makes such undesirable metabolic processes unlikely. Moreover, the quick binding assay at ice temperature, as it was
used by us, prevents possible receptor and hormone degradation and modiWcation by hazardous enzymatic activities.
Although we have presented only one example of an
E. coli strain expressing a plant membrane receptor, the
method has good potential to be applied successfully with
various eukaryotic receptors and bacteria. Recently, we
used this method to analyze the properties of another cytokinin receptor, AHK3 (unpublished results). It should be
noted that prokaryotes have receptors that are related to
the histidine kinase receptors studied by us. This might be
advantageous for this type of analysis. However, the level
of AHK receptor expression in the E. coli strain was rather
moderate; the corresponding band could not be detected in
a standard Coomassie-stained gel separating total bacterial
proteins (data not shown). According to our estimation, the
average level of CRE1/AHK4 expression was equal to 0.5
to 1.2 £ 105 protein molecules per bacterial cell. Taking into
account that the binding aYnity of the analyzed CRE1/
AHK4 cytokinin receptor (Kd D 2–6 nM) is one to two
orders of magnitude lower than the binding aYnities of
some animal hormone–receptor interactions (Kd can be in
the picomolar range [11]), far fewer bacterial cells will be
needed to assess reliable binding in the case of eVectively
expressed receptor possessing higher aYnity to its ligand.
It is also clear that proper processing of the protein and
correct insertion into the E. coli membrane are required for
successful experiments. A possible drawback of the method
is that in some cases certain posttranslational modiWcations
that might be required for proper functioning (e.g., glycosylation) are not carried out by the E. coli cell. Nevertheless,
in our opinion, the advantages outlined above outweigh
these possible limitations when using this method.
Acknowledgments
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Sfb 449), the Russian Foundation of Basic Research (NN 01-04-04002/03-04-04001/0404-49120), and INTAS (N 01-0602).
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