A novel catalytic function of synthetic IgG -binding domain (Z Domain) from staphylococcal protein a: Light emission with coelenterazine

Article abstract of DOI:10.1111/php.12192

The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-utilizing luciferase. The Z domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence properties were characterized by comparing with coelenterazine-utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z-gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG-binding region of the Z domain. The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-utilizing luciferase. The catalytic properties of the Z domain and the dimmer of Z domain are close to the Oplophorus luciferase and Gaussia luciferase, respectively.

Full text of DOI:10.1111/php.12192

Received Date : 25-Aug-2013
Accepted Date : 15-Oct-2013
Article type : Research Article
A novel catalytic function of synthetic IgG-binding domain (Z
domain) from staphylococcal protein A: Light emission with
coelenterazine
Satoshi Inouye* and Yuiko Sahara-Miura
Yokohama Research Center, JNC Co., Kanazawa, Yokohama 236-8605, Japan
* Corresponding Author: E-mail: Tel: 81-45-786-5502 / Fax: 81-45-786-5512;
(sinouye@jnc-corp.co.jp)
Abstract
The synthetic IgG-binding domain (Z domain) of staphylococcal protein A has a catalytic
function to oxidize coelenterazine to emit light like a coelenterazine-utilizing luciferase. The Z
domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence
properties were characterized by comparing with coelenterazine-utilizing luciferases,
including Renilla luciferase, Gaussia luciferase and the catalytic domain of Oplophorus
luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and
the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys
(36.8%) > Z-domain (1.1%) > bovine serum albumin (0.9%) > staphylococcal protein A (0.1%)
and the background value of coelenterazine (0.1%) in our conditions. The luminescence
properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the
luminescence pattern, the emission spectrum, the stimulation by halogen ions and non-ionic
detergents, and the substrate specificity for coelenterazine analogues. In contrast, the
luminescence properties of Z-gCys were close to the catalytic domain of Oplophorus luciferase.
The catalytic region of the Z domain for the luminescence reaction might be different from the
IgG-binding region of the Z domain.
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Abbreviations: ZZ-gCys, ZZ domain with a cysteine residue at the carboxyl terminus; Z-gCys,
Z domain with a cysteine residue at the carboxyl terminus; Z-domain, a monomer of Z domain;
MCS, multiple cloning site; Imax, initial maximum intensity of luminescence: rlu, relative light
units; PBS, 10 mM phosphate buffer (pH 7.4) containing 137 mM NaCl and 2.7 mM KCl.
Introduction
Protein A of Staphylococcus aureus has a high specific affinity to immunoglobulin G
(IgG) and binds the Fc region of IgG from most mammalian species (1). Protein A has been
used in the IgG purification and the immunological assay (1). The gene fragment coding for a
functional analogue of IgG-binding domain B of protein A, designated Z domain (58 amino
acid residues), was chemically synthesized and expressed in Escherichia coli cells (2). The ZZ
domain consisting of two repeat sequences of the Z domain with 116 amino acid residues
shows the binding ability to IgG (2) and was used as an affinity tag for protein purification (3).
Recently, we used the ZZ domain as a soluble partner in the cold inducible expression
system of E. coli cells (4, 5) and succeeded in expressing the heterogeneous proteins efficiently
as a soluble form in the cytoplasm of E. coli cells by using a novel expression vector of
pCold-ZZ-X (4). To study the formation of the fluorescent chromophore possessing an
imidazolone structure in green fluorescent protein (GFP) (6-9), we expressed the fusion protein
of the ZZ domain to GFP (ZZ-GFP) at 15 oC in E. coli cells and 139 mg of the purified ZZ-GFP
was obtained from 2 L of cultured cells (unpublished). In the course of characterizing the
biochemical properties of ZZ-GFP, we found that ZZ-GFP has a catalytic function like a
luciferase with coelenterazine (a luciferin). Previously, we purified recombinant GFP from E.
coli cells (6,7) and examined the luminescence activity of GFP with coelenterazine under
various assay conditions. However, GFP did not show any luminescence activity with
coelenterazine (unpublished). From these results, we presumed that the ZZ domain of ZZ-GFP
could catalyze the oxidation of coelenterazine to emit light in the following reaction scheme.
OH
OH
O
O
N
N
N
NH
ZZ-domain
+ O2
+ CO2 + Light
N
H
N
HO
HO
Coelenterazine
Coelenteramide
Coelenterazine, an imidazopyrazinone (3,7-dihydropyrazinon-3-one) compound, is
widely distributed in marine organisms (10,11) and is known as a luciferin for many luciferases
including Renilla (12), Oplophorus (13), Periphylla (14), Gaussia (15), Metridia (16) and
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Conchoecia (17) (referred to as “coelenterazine-type luciferase” in this paper). Coelenterazine
also serves as a light–emitting substrate for the Ca2+-binding photoproteins such as aequorin
(18, 19).
On the other hand, we have prepared recombinant aequorin and Gaussia luciferase having
a reactive cysteine residue to conjugate with a maleimide-activated ligand and used them for
bioluminescent immunoassays (20, 21). In the similar way, we prepared the ZZ domain
possessing a reactive cysteine residue at the carboxyl terminus, designated ZZ-gCys, and used
ZZ-gCys as an affinity ligand to purify IgG (unpublished). Under these circumstances, we
examined the luminescence activity of ZZ-gCys with coelenterazine and found that the ZZ
domain catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-type
luciferase (12-17).
In this report, we purified the Z domain derivatives, ZZ-gCys, Z-gCys and Z-domain and
characterized their luminescence properties by comparing with those of coelenterazine-type
luciferases. As a result, we concluded that the catalytic properties of ZZ-gCys and Z-gCys are
similar to that of Gaussia luciferase (22) and Oplophorus luciferase (23), respectively, and a
small protein/polypeptide can catalyze the oxidation of coelenterazine.
MATERIALS AND METHODS
Construction of the expression vectors for ZZ domain (ZZ-gCys) and Z domains
(Z-gCys and Z-domain) in E. coli cells. The strategy for constructing the expression vectors
was summarized in Fig. 1. The cold inducible expression vectors of pCold-ZZ-X (4) and
>Figure 1<
pCold-ZZ (4) were used for the ZZ domain (ZZ-gCys, the ZZ domain possessing a
flexible linker and a cysteine residue at the carboxyl terminus) and the Z domains (Z-gCys and
Z-domain), respectively. The expression vector of pCold-ZZ-X (4) has the sequences of a
six-histidine tag for the nickel chelate affinity purification, the ZZ domain (116 amino acid
residues), and the multiple cloning site (MCS). The protein expression was under the control
of the cspA promoter and the lac repressor from a pCold II vector (24). To express ZZ-gCys,
the oligonucleotide for a flexible linker sequence with a cysteine residue
(GGGGSGGGGSGGGGC: gCys) was chemically synthesized and inserted into MCS of
pCold-ZZ-X as follows: the synthetic oligonucleotides of gCys Linker-F (5’AG CTT GGT
GGT GGT GGT TCT GGT GGT GGT GGT TCT GGT GGT GGT GGT TGC TAA GCT AGC
CTG CA 3’, new NheI site underlined) and gCys Linker-R (5’ G GCT AGC TTA GCAACC
ACC ACC ACC AGAACC ACC ACC ACC AGAACC ACC ACC ACC A 3’) for “gCys
Linker” were synthesized (Fig. 1) and inserted into the HindIII/PstI site of pCold-ZZ-X to give
pCold-ZZ-gCys (Fig. 2-A). The expressed ZZ-gCys had the sequences of histidine-tag, ZZ
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domain, followed by the flexible linker with a new cysteine residue at the carboxyl terminus.
For expressing Z-gCys and Z-domain in E. coli cells, the vectors of pCold-Z-Cys and pCold-Z
were prepared by self-ligation after digesting with BglII (Fig. 1).
Expression and purification of ZZ domain (ZZ-gCys) and Z domains (Z-gCys and
Z-domain) from E. coli cells. The bacterial strain BL21 (Novagen, Madison, WI) was used as
a host. The procedures for the protein expression were essentially the same as previously
described (4,5). Briefly, the seed culture of BL21 possessing an expression vector was grown
in 10 mL of Luria-Bertani broth containing ampicillin (100 μg/mL) at 37 oC for 18 h, and was
transferred into 400 mL of LB broth containing 20 μL of antifoam (Disfoam CE457: NOF Co.,
Tokyo, Japan) in a 3 L of Sakaguchi flask, incubated for 2.5 h at 37 oC and then cooled on an
ice-water bath for 1 h. After adding isopropyl-β-thiogalactopyranoside (Wako Pure Chemicals,
Osaka, Japan) to the culture medium at a final concentration of 0.2 mM, the bacterial cells were
incubated at 15 oC for 17 h. The cells harvested by centrifugation at 4,700g for 5 min were
suspended in 200 mL of 50 mM Tris-HCl (pH 7.6) and were disrupted by sonication using a
Brabson model 250 sonifire (Danbury, CT) for 9 min (3 min x 3) in an ice-water bath. After
centrifugation at 12,000g for 20 min at 4 oC, the supernatant obtained was applied on an
IgG-Sepharose 6 Fast Flow column (φ 2.5 x 6.0 cm and φ1.5 x 6.5 cm for 2 L and 0.8 L of
cultured cells, respectively: GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM
Tris-HCl (pH 7.4) containing 150 mM NaCl and 0.5% (w/v) Tween 20 (TBST) at room
temperature. After washing the column with 300 mL of TBST and 100 mL of ammonium
acetate (pH 5.0), the adsorbed proteins were eluted by stepwise with 10 mL of 0.5 M acetic acid
(pH 3.4). The eluted fraction was analyzed by SDS-PAGE and the protein fractions were
dialyzed against 4 L of 100 mM sodium phosphate buffer (pH 7.3) at 4 oC for 72 h. For
long-term storage, the protein dialyzed was stored at -80 oC.
Determination of luminescence activity. Recombinant protein A (rPA50, Lot.
RN092399) was obtained from RepliGen Co. (Waltham, MA). Bovine serum albumin
(Fraction V: A3059-100G, Lot. 116K0757) and human IgG from human serum (Lot.
44204601) were obtained from Sigma Co. (St. Louis, MO) and Oriental Yeast Co. Ltd. (Tokyo,
Japan), respectively. Recombinant Renilla luciferase (25) and Gaussia luciferase (22) was
purified as previously described. Coelenterazine was obtained form JNC Corp. (Tokyo, Japan)
and the C-2 modified coelenterazine analogues were prepared as previously described (26).
(S)-Cypridina luciferin was obtained from Prolume Ltd. (Pinetop, AZ). The reaction mixture
(100 μL) contained a luciferin (coelenterazine, its analogue, or Cypridina luciferin;1 μg
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dissolved in 1 μL of ethanol) in 30 mM Tris-HCl (pH 7.6)–10 mM EDTA and the
luminescence reaction was initiated by the addition of 1-15 μL of a protein solution into the
reaction mixture. The luminescence intensity (n = 2) was measured using an Atto (Tokyo,
Japan) AB2200 luminometer (Ver.2.07, rev.4.21) equipped with a Hamamatsu R4220P
photomultiplier in 0.1 s intervals for 60 s without an attenuation filter. As recombinant
aequorin shows 4.8 x 1015 photons/mg protein (27), we used recombinant aequorin as a light
standard for the luminometer. The initial maximum intensity of luminescence (Imax) of 1 pg of
the purified recombinant aequorin (27) showed 1.3 x 103 relative light unit (rlu)/0.1 s and one
rlu was estimated to be 3.7 x 103 photons/0.1 s in our assay conditions. To examine the effects
of NaCl concentrations and halogen ions on luminescence activity, 50 mM sodium phosphate
buffer (pH 7.5) was used instead of 100 mM Tris-HCl (pH 7.6). For determining the optimal
pH, the following buffers were used: 100 mM sodium acetate buffer (pH 5.0), 100 mM sodium
phosphate buffer (pH 6.0, 6.5, 7.0 and 7.5) and 100 mM Tris-HCl buffer (pH 7.0, 7.5, 8.0 and
8.5).
Protein analysis. Protein concentration was determined by the dye-binding method of
Bradford (28) using a commercially available kit (Bio-Rad, Richmond, CA) and bovine serum
albumin as a standard (Pierce; Rockford, IL). The concentration of purified ZZ-gCys, Z-gCys
and Z-domain was determined by the absorbance at 280 nm in 0.1% solution. The calculated
absorbance values of 0.166, 0.131 and 0.1500, which were obtained using a program
ProtParam (http://web.expasy.org/protparam/), were used for ZZ-gCys, Z-gCys and Z-domain.
SDS-PAGE analysis was carried out under reducing or non-reducing conditions using a 16%
separation gel (TEFCO, Tokyo, Japan) (29) and the gels were stained with a colloidal CBB
staining kit (TEFCO).
Measurement of luminescence emission spectrum. The luminescence emission spectra
of ZZ-gCys, Z-gCys and Z-domain with coelenterazine were measured on a Jasco FP-6500
fluorescence spectrophotometer (emission band width, 20 nm; response, 0.5 sec; sensitivity,
medium; scan speed, 1000 nm/min) at 22~25 oC with the excitation light source turned off.
The reaction mixture (500 μL) contained 0.5 mg of the purified proteins in 30 mM Tris-HCl
(pH 7.6)-10 mM EDTA and the luminescence reaction was initiated by the addition of 5 μg of
coelenterazine dissolved in 5 μL of ethanol. The corrected luminescence emission spectrum
was obtained according to the manufacture’s protocol.
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Identification of oxidation products of coelenterazine by HPLC analysis. The reaction
mixture (0.5 mL) contained 0.5 mg of ZZ-gCys and 10 μg of coelenterazine (dissolved in 10
μL of ethanol) in 30 mM Tris-HCl (pH 7.6)-10 mM EDTA. After the luminescence reaction
for 1 min at 25 oC, 100 μL of the reaction mixture was added to 0.5 mL of ether and stored at
-80 oC over 10 min. The ether layer recovered by centrifugation at 10,000 rpm for 3 min was
dry down in vacuo. The resultant precipitate was dissolved in 20 μL of ethanol and 5 μL was
analyzed by HPLC. The analytical conditions were follows: Agilent (CA, USA) 1100 series
HPLC system. column: Wakosil 5C4 (5 mm, 4.6 mm × 250 mm, Wako Pure Chemicals);
eluting solvent: CH3CN containing 0.1% trifluoroacetic acid; gradient elution: 40% CH3CN
for 10 min, 40-50% CH3CN for 20 min, 50-80% CH3CN for 10 min and 80% CH3CN for 10
min; flow rate: 0.5 mL/min; detector: 225 nm, 330 nm and 450 nm. The retention times of
coelenterazine, coelenteramide, and coelenteramine were 14.3 min, 21.0 min, 13.6 min,
respectively. The amounts of coelenterazine, coelenteramide, and coelenteramine were
determined by using the authentic solutions of synthetic compounds (30).
RESULTS AND DISCUSSION
Expression and purification of ZZ-gCys, Z-gCys and Z-domain. The ZZ-gCys, Z-gCys
and Z-domain were expressed in E. coli cells using the cold inducible expression vectors of
pCold-ZZ-gCys, pCold-Z-gCys and pCold-Z, respectively, (Fig. 1), and were purified from the
soluble fractions of E. coli cells in a single step using an IgG Sepharose column. As the
amounts of the purified Z-gCys and Z-domain were not determined accurately by the
dye-binding method of Bradford (28), the amount of protein was estimated by using the
calculated absorbance value at 280 nm in 0.1% solution as described in Materials and Methods.
The yield of the purified ZZ-gCys was 96.4 mg from 2 L of cultured cells, which was in good
agreement with that by the dye-binding method. From 0.8 L of cultured cells, the yields of
Z-gCys and Z-domain estimated were 29.4 mg and 21.1 mg, respectively (Table 1). On
SDS-PAGE analysis under reducing conditions, the single bands of ZZ-gCys (19 kDa), Z-gCys
(11 kDa) and Z-domain (10.5 kDa) were observed and the purities were estimated to be over
95% (Fig. 3A). Under non-reducing conditions, however, the dimer of Z-gCys with a disulfide
bond was detected at the molecular size of 23.5 kDa, showing over 50% density on a gel (Fig.
3B, lane 2). On the other hand, the percentage of the ZZ-gCys dimer was lower than that of
Z-gCys. The density of ZZ-gCys dimer (32.5 kDa) on a gel was ~10% of the purified ZZ-gCys.
The dimer formation of ZZ-gCys increased slowly at 4 oC over 1 month (Fig. 3B, lane 1).
From this reason, we used the protein fractions containing a dimer for assays.
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Luminescence activity of ZZ-gCys, Z-gCys and Z-domain. The luminescence activities
of ZZ-gCys, Z-gCys and Z-domain with coelenterazine as a substrate were examined by
comparison with recombinant staphylococcal protein A, bovine serum albumin (BSA) and
recombinant Renilla luciferase. As shown in Table 2, ZZ-gCys and Z-gCys showed
luminescence activity with coelenterazine and Z-domain had measurable luminescence activity.
In contrast, protein A did not show the luminescence activity and the initial maximum intensity
of luminescence (Imax) was same to that of the background level from coelenterazine (Table 2).
BSA had very weak luminescence activity with coelenterazine as previously reported (31,32),
but was only 0.9% of Imax to ZZ-gCys. The Imax value of ZZ-gCys was 2.7 times higher than
that of Z-gCys. The integrated luminescence activity of ZZ-gCys for 60 s was 5.6 times lower
than that of Z-gCys, due to the different luminescence pattern from Z-gCys (Fig. 4). The order
of Imax was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin
(0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%).
The dimer of Z-gCys gave the higher luminescence activity than that of the monomer of
Z-domain. The luminescence intensities of ZZ-gCys and Z-gCys were dependent on the
amounts of proteins (Table 2). Further, the luminescence activity of ZZ-gCys was lost by heat
treatment at 95 oC for 10 min (Table 3). Also Z-gCys and Z-domain lost luminescence activity
by heat treatment (data not shown). These results indicated that the Z domain has a catalytic
function to emit light and the light-emitting process with coelenterazine is an enzymatic
reaction like a coelenterazine-type luciferase. However, the Imax value of ZZ-gCys was over
60,000 times lower than that of recombinant Renilla luciferase (25) (Table 2). On the other
hand, the Imax value of Renilla luciferase was ~10 times lower than that of Gaussia luciferase
and was ~100 times higher than that of the catalytic 19 kDa domain of Oplophorus luciferase
as previously reported [33].
Oxidation process of coelenterazine by ZZ-gCys. To investigate the oxidation process of
coelenterazine by ZZ-gCys, the luminescence products of coelenterazine were analyzed by
reversed-phase HPLC. The reaction mixture containing 0.5 mg of ZZ-gCys and 10 μg of
coelenterazine was incubated only for 1 min in 30 mM Tris-HCl (pH 7.6)-10 mM EDTA,
avoiding the auto-oxidation of coelenterazine. The luminescence products of coelenterazine
were identified as coelenteramide and coelenteramine, and the reaction mixture contained
unreacted coelenterazine (88%), coelenteramide (5%) and coelenteramine (7%). The catalytic
ability of luminescence by ZZ-gCys was low, but it was interesting that the value of 7% for
coelenteramine was unexpectedly high. As coelenteramine was not hydrolyzed from
coelenteramide in our reaction conditions, coelenteramine might be produced directly from
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coelenterazine after the oxygenation of coelenterazine [33]. Presumably, the low luminescence
activity of the Z and ZZ domains might be explained by low oxidation efficiency of
coelenterazine and less stabilization of the light-emitting species of coelenteramide.
Luminescence patterns and luminescence emission spectra of ZZ-gCys, Z-gCys and
Z-domain with coelenterazine. The luminescence pattern of a luciferin-luciferase reaction
gives an insight into the kinetics property of a luciferase, as previously described in the case of
the firefly luciferase-luciferin reaction (34). The coelenterazine-type luciferases from Renilla
(25), Oplophorus (23) and Conchoecia (17) show the steady-state kinetics with a glow
luminescence pattern. In contrast, Gaussia and Periphylla luciferases show a rapid decay
pattern with flash-light emission in the presence of excess coelenterazine (14, 15, 35). As
shown in Fig. 4A, light emission by ZZ-gCys rose quickly followed by a rapid decay, and then
continuous weak-glow luminescence was observed. This luminescence pattern was similar to
that of Gaussia luciferase and Periphylla luciferase. The rapid decay of luminescence in these
proteins might be explained by the product inhibition of coelenteramide, the oxidation product
of coelenterazine. On the other hand, Z-gCys and Z-domain showed the continuous glow
luminescence, similar to Renilla, Oplophorus and Conchoecia luciferases (Fig. 4A).
The luminescence emission spectra of ZZ-gCys, Z-gCys and Z-domain with
coelenterazine were measured under the same conditions at pH 7.6, and the corrected emission
peaks and a full width at half maximum of spectra (FWHM) were determined (Fig. 5). The
emission peaks of ZZ-gCys, Z-gCys and Z-domain were at 490 nm (FWHM = 80 nm), 460 nm
(FWHM = 77 nm) and 466 nm (FWHM= 83 nm), respectively. The luminescence emission
spectrum of ZZ-gCys was different from that of Z-gCys and Z-domain. Interestingly, the
emission peak and FWHM of ZZ-gCys was close to that of Gaussia luciferase (λmax = 488 nm,
FWHM = 81 nm) (22). In contrast, the luminescence emission spectra of Z-gCys and
Z-domain were close to that of the catalytic 19 kDa domain of Oplophorus luciferase (λmax =
460 nm, FWHM = 72 nm) (23). The different luminescence emission spectra of them were
caused by the emitting species of coelenteramide, the oxidation product of coelenterazine. To
understand the luminescence mechanism of coelenterazine and Cypridina luciferin by a
luciferase, the studies on the chemiluminescence of imidazopyrazinone derivatives have been
performed in organic solvents (10, 36, 37). The light-emitting species of coelenteramide have
been proposed by the fluorescence spectral analyses. In our findings, the different
light-emitting spectra between the Z domain and the ZZ domain might be explained by the
light-emitting species of coelenteramide; amide anion of coelenteramide (λmax 450 - 470 nm)
for Z domain and phenolate anion (λmax 480 nm) for ZZ domain. However, it is difficult to
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explain how difference spectra were produced by an identical domain in the Z domain and the
ZZ domain.
Effects of IgG binding to Z domain on luminescence activity. It is known that the Z
domain has an ability to bind IgG (1, 2) and the Z domain derivatives were purified by an IgG
Sepharose column. To investigate whether the binding of IgG to the Z domain inhibits
luminescence activity of ZZ-gCys, Z-gCys and Z-domain, the effect of IgG on luminescence
activity was examined (Table 4). If the catalytic region of the Z domain with coelenterazine
might be identical to the IgG-binding region, the luminescence activity should be decreased
significantly. However, the addition of IgG to ZZ-gCys at a 1:1 molar ratio gave about 1.5 time
stimulation of luminescence intensity (Fig. 4C), and the luminescence activity of Z-gCys was
not stimulated or inhibited. These results suggested that the catalytic region of the Z domain
with coelenterazine was different from the IgG-binding region of the Z domain. Previously, the
solution structures of the B and Z domains of staphylococcal protein A have been determined
by NMR spectroscopy (38-41). These domains are composed of an antiparallel three α-helices
(helix 1, helix 2 and helix 3) and the Fc portion of IgG binds the helix 1 and helix 2 in the B and
Z domains. The luminescence stimulation of ZZ-gCys by IgG could be explained by binding
IgG to the helix 1 and helix 2 of Z domain and the protein structure of ZZ-gCys could be
stabilized. Presumably, a part of the helix 3 of the Z domain was related to the luminescence
reaction. On the other hand, the stimulation of luminescence intensity was also observed in the
presence of BSA, and might be explained by the protein stabilization (Table 4).
Optimal pH for the luminescence reaction by ZZ-gCys and Z-gCys with coelenterazine.
The luminescence activity (Imax) of ZZ-gCys and Z-gCys was determined under various pH
conditions. The optimal pH for ZZ-gCys was found to be around 7.5 in 100 mM Tris-HCl
buffer, and over 80% activity was observed between pH 7.0 and 8.0. Thus, the pH profile of
ZZ-gCys was close to that of Gaussia luciferase (22). On the other hand, Z-gCys showed the
optimal pH at 8.0 in sodium phosphate buffer and Tris-HCl buffer, and over 80% activity was
observed between pH 7.0 and 9.0 (Supplementary Fig. S1) .
Stimulation of luminescence activity of ZZ-gCys by halogen ions and non-ionic
detergent. Previously, we reported that the luminescence intensity of recombinant Gaussia
luciferase was stimulated by non-ionic detergents, sodium chloride, and halogen ions (22). To
compare the luminescence property of ZZ-gCys with Gaussia luciferase, the effects of Tween
20, sodium chloride and halogen ions on the luminescence activity of ZZ-gCys were examined.
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The Imax value of ZZ-gCys was 4-folds stimulated by 0.01% Tween 20 (Fig. 4B and Fig. 6A).
The effective concentration of 0.01% Tween 20 on ZZ-gCys was similar to that of Gaussia
luciferase. Further, 100 mM NaCl stimulated the luminescence intensity of ZZ-gCys
significantly (Fig. 6B, Table 5). Further, the halogen ions such as Cl-, Br-, and I- played as a
stimulator on the luminescence activity of ZZ-gCys (Table 6). The stimulation of halogen ions
was the same to Gaussia luciferase (22) and the mechanism of stimulation was not clear. In
contrast, the luminescence intensities of Z-gCys and Z-domain were not stimulated by Tween
20 and halogen ions. These results strongly suggested that the catalytic process of
luminescence by ZZ-gCys might be similar to that by Gaussia luciferase.
Substrate specificities of ZZ-gCys, Z-gCys and Z-domain for coelenterazine analogues
and Cypridina luciferin. The substrate specificities of ZZ-gCys, Z-gCys and Z-domain were
characterized using the C-2 modified coelenterazine analogues (26) and were compared with
the coelenterazine-type luciferases (33). As a result, ZZ-gCys showed narrow substrate
specificity, indicating highly specific for coelenterazine (Table 7). This narrow substrate
specificity of ZZ-gCys was similar to that of Gaussia luciferase, as previously reported (22, 33).
On the other hand, Z-gCys and Z-domain had broad specificity for coelenterazine analogues
and this was similar to Oplophorus luciferase (23,25, 33). Among coelenterazine analogues,
ameh-coelenterazine is only used for the catalytic19 kDa protein of Oplophorus luciferase (33),
but not for Renilla luciferase, Gaussia luciferase and aequorin (33). Other
imidazopyrazinone-type luciferin, (S)-Cypridina luciferin, was not utilized for the
luminescence reactions by ZZ-gCys, Z-gCys and Z-domain, as same as by Gaussia luciferase
and Oplophorus luciferase.
Similarity of the luminescence reaction of the Z domain to coelenterazine-type luciferase. It
is known that coelenterazine-type luciferases from Renilla, Oplophorus and Gaussia do not
have any similarities in their primary structures. The luminescence mechanism of
coelenterazine catalyzed by coelenterazine-type luciferases is essentially the same (10). The
differences with luminescence efficiency and emission spectrum might be explained by the
environment of the light-emitter species in a protein (enzyme) (36, 37). As we described above,
the Z domain and the ZZ domain clearly showed luminescence activity with coelenterazine.
The primary structure of the Z domain with 58 amino acid residues has no significant
homology with that of coelenterazine-type luciferases. However, the luminescence properties
of the ZZ domain and the Z domain were close to Gaussia luciferase and Oplophorus luciferase,
respectively. The secreted Gaussia luciferase consists of 168 amino acid residues, having two
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repeat domain with 71 amino acid residues (22), and each domain in Gaussia luciferase shows
the weak luminescence activity of luciferase. The catalytic domain of Oplophorus luciferase is
169 amino acid residues and composes of two structural units (23). These results suggested
that a small protein/polypeptide could have a catalytic ability to oxidize coelenterazine and the
folded structure of a luciferase is of considerable important to a catalytic function of
luminescence.
Conclusion. We found that the synthetic IgG-binding domains (ZZ domain and Z
domain) derived from staphylococcal protein A have a catalytic function to oxidize
coelenterazine to emit light like a coelenterazine-utilizing luciferase. The ZZ domain showed
the similar luminescence properties to Gaussia luciferase, including the luminescence pattern,
the emission spectrum, the stimulation by halogen ions and non-ionic detergents, and the
substrate specificity for coelenterazine analogues. On the other hand, the luminescence
properties of the Z domain were close to the catalytic domain of Oplophorus luciferase. The
catalytic region for the luminescence reaction might be different from the IgG-binding region
of the Z domain.
Acknowledgement
The authors thank Dr. T. Hosoya for helpful discussions.
Supplementary Material:
Figure S1 can be found online on DOI: xxxxx
References
1. Langone, J. J. (1982) Protein A of Staphylococcus aureus and related immunoglobulin
receptors produced by streptococci and pneumonococci. Adv. Immunol. 32, 157-252.
2. Nilsson, B., Moks, T., Jansson, B., Abrahmsén, L., Elmblad, A., Holmgren, E., Henrichson, C.,
Jones, T. A. and Uhlén M. (1987) A synthetic IgG-binding domain based on staphylococcal
protein A. Protein Eng. 1, 107-13.
3. Samuelsson, E., Wadensten, H., Hartmanis, M., Moks, T. and Uhlén, M. (1991) Facilitated in
vitro refolding of human recombinant insulin-like growth factor I using a solubilizing fusion
partner. Bio/Technology 9, 363-366
4. Inouye, S. and Sahara, Y. (2008) Soluble protein expression in E. coli cells using IgG binding
domain of protein A as a solubilizing partner in the cold induced system, Biochem. Biophys.
This article is protected by copyright. All rights reserved.

Res. Commun. 376, 448-453.
5. Inouye, S. and Sahara, Y. (2009) Expression and purification of the calcium binding
photoprotein mitrocomin using ZZ-domain as a soluble partner in E. coli cells. Protein
Express. Purif. 66, 52-57.
6. Inouye, S. and Tsuji, F.I. (1994) Aequorea green fluorescent protein: Expression of the gene
and fluorescence characteristics of the recombinant protein. FEBS Lett. 341, 277-280.
7. Inouye, S. and Tsuji, F.I. (1994) Evidence for redox forms of the Aequorea green fluorescent
protein. FEBS Lett. 351, 211-214.
8. Ogawa, H., Inouye, S., Tsuji, F.I., Yasuda, K. and Umesono, K. (1995) Localization,
trafficking, and temperature-dependence of the Aequorea green fluorescent protein in
cultured vertebrate cells. Proc. Natl. Acad. Sci. USA. 92, 11899-11903.
9. Heim, R., Prasher, D. C. and Tsien, R. Y. (1994) Wavelength mutations and
posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA.
91,12501-12504.
10. Teranishi, K. (2007) Luminescence of imidazo(1,2-a)pyrazin-3(7H)-one compounds,
Bioorg. Chem. 35, 82–111.
11. Thomson, C. M., Herring, P. J., and Campbell, A. K. (1997) The widespread
occurrence and tissue distribution of the imidazopyrazine luciferin. J. Biolumi. Chemilumi.
12, 87-91.
12. Lorenz, W.W., McCann, R.O., Longiaru, M. and Cormier, M.J. (1991) Isolation and
expression of a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. USA. 88,
4438-4442.
13. Inouye, S., Watanabe, K., Nakamura, H. and Shimomura, O. (2000) Secretional luciferase of
the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone
luciferase. FEBS Lett. 481, 19-25.
14. Shimomura, O., Flood, Per R., Inouye, S. , Bryan, B. and Shimomura, A. (2001) Isolation and
properties of the luciferase stored in the ovary of the scyphozoan medusa Periphylla
Periphylla. Biol. Bull. 201, 339-347.
15. Verhaegen, M. and Christopoulos, T.K. (2002) Recombinant Gaussia luciferase.
Overexpression, purification, and analytical application of a bioluminescent reporter for
DNA hybridization. Anal. Chem. 74, 4378-4385.
16. Markova, S. V., Golz, S., Frank, L. A., Kalthof, B. and Vysotski, E. (2004) Cloning and
expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel
secreted bioluminescent reporter enzyme. J. Biol. Chem. 279, 3212-3217.
17. Oba, Y., Tsuduki, H., Kato, S., Ojika, M. and Inouye, S. (2004) Identification of
This article is protected by copyright. All rights reserved.

luciferin-luciferase system and quantification of coelenterazine by mass
spectrometry in the deep-sea luminous ostracod, Conchoecia pseudodiscophora.
ChemBioChem. 5, 1495-1499.
18. Head, J.F., Inouye, S., Teranishi, K. and Shimomura, O. (2000) The crystal structure of the
photoprotein aequorin at 2.3Åresolution. Nature 405, 372-376.
19. Toma, S., Chong, K.T., Nakagawa, A., Teranishi, K., Inouye, S., and Shimomura, O. (2005)
The crystal structures of semi-synthetic aequorins. Protein Sci. 14, 409-416.
20. Inouye, S. and Sato, J. (2008) Recombinant aequorin with a reactive cysteine residue for
conjugation with maleimide-activated antibody. Anal. Biochem. 378, 105-107.
21. Inouye, S., Sato, J. and Sahara-Miura, Y. (2011) Recombinant Gaussia luciferase with a
reactive cysteine residue for chemical conjugation: expression, purification and its
application for bioluminescent immunoassays. Biochem. Biophys. Res. Commun. 410,
792-797.
22. Inouye, S. and Sahara, Y. (2008) Identification of two catalytic domains in a luciferase
secreted by the copepod Gaussia princeps. Biochem. Biophys. Res. Commun. 365, 96-101.
23. Inouye, S. and Sasaki, S. (2007) Overexpression, purification and characterization of the
catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus
gracilirostris. Protein Express. Purif. 56, 261-268
24. Qing, G., Ma, L-.C., Khorchid, A., Swapna, G.V.T., Mal, T.K., Takayama, M.M., Xia, B.,
Phadtara, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M. and Inouye, M. (2004)
Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22,
877-882.
25. Inouye, S. and Shimomura, O. (1997) The use of Renilla luciferase, Oplophorus luciferase
and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine
analogues as substrate. Biochem. Biophys. Res. Commun. 233, 349-353.
26. Inouye, S., Iimori, R. Sahara, Y., Hisada, S. and Hosoya, T. (2010) Application of new
semisynthetic aequorins with long half-decay time of luminescence to G-protein-coupled
receptor assay. Anal. Biochem. 407, 247-52.
27. Shimomura, O. and Inouye, S. (1999) The in situ regeneration and extraction of recombinant
aequorin from Escherichia coli cells and the purification of extracted aequorin. Protein Expr.
Purif. 16, 91-95.
28. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,
248-254.
This article is protected by copyright. All rights reserved.

29. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680-685.
30. Inouye, S. and Hosoya, T. (2009) Reconstitution of blue fluorescent protein from
recombinant apoaequorin and synthetic coelenteramide. Biochem. Biophys. Res.
Commun. 386, 617–622.
31. Zhao, H., Doyle, T. C., Wong, R.J., Cao, Y., Stevenson, D. K., Piwnica-Worms, D. and
Contag, C. H. (2004) Characterization of coelenterazine analogs for measurements of
Renilla luciferase activity in live cells and living animals. Mol. Imaging 3, 43-54.
32. Keyaerts, M., Heneweer, C., Gainkam, L. O., Caveliers, V., Beattie, B.J., Martens, G. A,
Vanhove, C., Bossuyt, A., Blasberg, R. G. and Lahoutte, T. (2011) Plasma protein binding of
luciferase substrates influences sensitivity and accuracy of bioluminescence imaging. Mol
Imaging Biol. 13, 59-66.
33. Inouye, S., Sahara-Miura, Y., Sato, J., Iimori, R., Yoshida, S. and Hosoya, T. (2013)
Expression, purification and luminescence properties of coelenterazine-utilizing luciferases
from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified
coelenterazines. Protein Express. Purif. 88, 150-156.
34. Inouye, S. （2010）Firefly luciferase: an adenylate-forming enzyme for multicatalytic
functions. Mol. Cell. Life Sci. 67, 387-404.
35. Suzuki, T., Kondo, C., Kanamori, T. and Inouye, S. (2011) Video-rate bioluminescence
imaging of matrix metalloproteinase-2 secreted from a migrating cell. PLoS ONE 6(9),
e25243
36. Goto, T. (1968) Chemistry of bioluminescence, Pure Appl. Chem. 17, 421-441.
37. Shimomura, O. and Teranishi, K. (2000) Light-emitters involved in the luminescence of
coelenterazine. Luminescence, 15, 51-58.
38. Gouda, H. Torigoe, H., Saito, A., Sato, M., Arata, Y. and Shimada, I. (1992)
Three-dimensional solution structure of the B domain of staphylococcal protein A:
comparisons of the solution and crystal structures. Biochemistry 31, 9665-9672
39. Gouda, H., Shiraishi, M., Takahashi, H., Kato, K., Torigoe, H., Arata, Y. and Shimada,
I. (1998) NMR study of the interaction between the B domain of staphylococcal
protein A and the Fc portion of immunoglobulin G. Biochemistry 37, 129-36.
40. Tashiro, M., Tejero, R., Zimmerman, D. E., Celda, B., Nilsson, B. and Montelione, G. T.
(1997) High-resolution solution NMR structure of the Z domain of staphylococcal protein A.
J Mol Biol. 272, 573-590.
41. Cedergren, L, Andersson, R., Jansson, B., Uhlén, M. and Nilsson, B. (1993) Mutational
analysis of the interaction between staphylococcal protein A and human IgG1. Protein Eng. 6,
This article is protected by copyright. All rights reserved.

441-448.
Table 1. Purification of ZZ-gCys, Z-gCys and Z-domain from E. coli cells using IgG
Sepharose column.
Total
volume
(mL)
Total
protein
(mg)
Protein
Purification steps
Soluble fraction (12,000g sup) from 2 L of
cultured cells
Dialysis fraction after IgG Sepharose
column (φ2.5 x 6 cm)
Soluble fraction (12,000g sup) from 0.8 L of
cultured cells
Dialysis fraction after IgG Sepharose
column (φ1.5 x 6.5 cm)
Soluble fraction (12,000g sup) from 0.8 L of
cultured cells
Dialysis fraction after IgG Sepharose
column (φ1.5 x 6.5 cm)
ZZ-gCys
200
22
80
19
80
10
880
94.6 a
384
Z-gCys
29.4 a
N.D.b
21.1 a
Z-domain
a Protein concentration is determined by the absorbance at 280 nm, as described in Materials
and Methods.
b N.D. Not determined.
Table 2. Luminescence activities of ZZ-gCys, Z-gCys and Z-domain using coelenterazine
as a substrate.
Luminescence intensity (rlu) b
Protein conc.
Protein
None
Integration
for 60 s
(μg/100 μL)
Imax
(0.1) a
(100) a
(0.8) a
(100) a
0
1
15
3,638
66,199
2,847
16,266
59,865
990
ZZ-gCys
5
456,992
2,632,829
251,305
2,565,133
8,536,031
4,721
15
1
(36.8) a
(561) a
Z-gCys
5
5,991
21,488
41
15
1
Z-domain
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(1.1) a
(0.1) a
(0.9) a
(5.9) a
(0.7) a
5
15
5
172
1,137
14
26,888
314,559
3,024
Protein A
100
5
28
8,350
(14.1) a
144
64,261
Bovine serum
albumin (BSA)
100
2,033
1,015,256
Renilla
luciferase
225,961,817 c
0.005
1,075,980
a
Relative luminescence intensity to ZZ-gCys (100% as 5 μg protein used).
b The reaction mixture (100 μL) contains 1 μg of coelenterazine (1 μg/μL dissolved in ethanol)
in 30 mM Tris-HCl (pH 7.6)-10 mM EDTA. The luminescence reaction is started by the
addition of the protein solution into the reaction mixture and luminescence intensity was
recorded by a luminometer for 60 s in 0.1 s intervals.
c
Integration for 30 s in 0.1 s intervals
.
Table 3. Effect of heat treatment on the luminescence activity of ZZ-gCys.
Heat
Luminescence intensity b
(Imax, rlu) (%)
20 (-)
Protein (5 μg/100 μL)
None
treatment a
-
-
+
-
+
-
+
13,572 (100)
366 (0.26)
14 (0)
ZZ-gCys
Protein A
BSA
14 (0)
144 (0.09)
71 (0.04)
a Protein solutions are heated at 95 oC for 10 min and used for assays.
b Assay conditions are the same as described in Table 2.
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Table 4. Effects of IgG and BSA on the luminescence activities of ZZ-gCys, Z-gCys and
Z-domain.
Protein molar
Luminescence intensity
Conditions a
ratio to IgG
or BSA
0:1
(Imax
,
rlu) (%)
IgG
38
BSA
0:1
711
ZZ-gCys
1:0
1:1
1:1
1:0
1:1
1:1
1:0
9,808 (100)
16,107 (164)
22,705 (231)
1,070 (100)
1,062 (100)
2,221 (208)
52 (100)
ZZ-gCys + IgG
ZZ-gCys + BSA
Z-gCys
Z-gCys + IgG
Z-gCys + BSA
Z-domain
Z-domain + IgG
Z-domain + BSA
1:1
1:1
138 (265)
840 (162)
a The reaction mixture (100 μL) contains IgG or BSA with Z-domain, Z-gCys or ZZ-gCys (5
μg) in PBS and incubate at room temperature for 20 min, and then the luminescence reaction
was started by the addition of 1 μg of coelenterazine (1 μg/μL dissolved in ethanol) into the
reaction mixture. The luminescence intensity was recorded by a luminometer.
Table 5. Effect of sodium chloride on the luminescence activities of ZZ-gCys, Z-gCys and
Z-domain.
Luminescence intensity (Imax, rlu) a
NaCl (mM)
ZZ-gCys
159
Z-gCys
9,949
Z-domain
199
0
100
28,620
9,495
339
a
The reaction mixture (100 μL) with or without 100 mM NaCl contains 1 μg of
coelenterazine in 50 mM sodiium phosphate buffer (pH 7.5) and the luminescence reaction is
started by the addition of ZZ-gCys, Z-gCys and Z-domian (10 μg protein).
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Table 6. Effect of halogen ions on the luminescence activity of ZZ-gCys.
Relative luminescence
Assay conditions (Salt conc. 50 mM)
intensity (Imax, %) a
100 mM Tris-HCl (pH 7.6)
50 mM Sodium phosphate (pH 7.5)
100.0
0.6
+ (NH4)2SO4
+ MgSO4
+ MgCl2
+ KF
11.2
9.8
86.4
0.7
+ NaF
+ KCl
+ NaCl
+ KBr
+ NaBr
+ KI
10.7
126.6
124.9
127.7
128.6
74.6
74.9
+ NaI
a
The reaction mixture (100 μL) contains 50 mM halogen ions and 1 μg of coelenterazine in 50
mM sodium phosphate buffer (pH 7.5) and the luminescence reaction is started by the addition
of ZZ-gCys (10 μg protein).
Table 7. Substrate specificities of ZZ-gCys, Z-gCys and Z-domain for coelenterazine
analogues.
Relative luminescence intensity (Imax, %) b
Coelenterazine
analogue a
Gaussia
Z
Z-gCys
ZZ-gCys
luciferase
100
0.03
0.04
0.8
Coelenterazine
100
64.3
45.1
49.9
116
100
68.0
54.2
43.2
136
100
0.1
0.1
0.7
5.3
7.0
1.4
0.6
0.7
0.6
7.8
bis-
hcp-
n-
h-
5.9
3iso-
3meo-
cf3-
i-
37.8
75.2
35.1
46.8
40.4
54.0
52.4
99.9
41.9
41.3
40.7
70.1
7.8
2.2
0.6
1.0
0.7
6.8
et-
meo-
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me-
3me-
ameh-
62.2
62.0
9.6
65.2
70.5
8.4
3.1
1.4
0.1
3.5
1.7
0.1
a Abbreviations of coelenterazine analogues are used from ref. 25 and 26.
b Assay conditions are the same as described in Table 2.
Figure legends
Fig. 1. A schematic representation of strategy for constructing the expression vectors for
ZZ-gCys, Z-gCys and Z-domain.
Z, IgG binding domain of the Z domain (58 amino acid residues); His-tag, six
histidine-tagged sequence for Ni-chelate affinity chromatography; MCS, a multiple cloning
site; gCys Linker, a flexible linker sequence with a cysteine residue (-SH) at the carboxyl
terminus (GGGGSGGGGSGGGGC).
Fig. 2. Expression vector for ZZ-gCys in E. coli cells.
A. Plasmid map of pCold-ZZ-gCys expression vector. cpsA, the promoter of cold shock
protein A; TEE, translational enhancing element; His-Tag, six histidine-tagged sequence; ZZ
domain, IgG binding domain (116 amino acid residues); gCys Linker, a flexible linker
sequence with a cysteine residue.
B. Amino acid sequence of the N- and C-terminal regions of ZZ-gCys.
Fig. 3. SDS-PAGE analyses of purified ZZ-gCys, Z-gCys and Z-domain. SDS-PAGE
analyses were performed under reducing conditions (A) and non-reducing conditions (B).
Lane 1, ZZ-gCys (5 μg); lane 2, Z-gCys (5 μg), lane 3, Z-domain (5 μg). The numbers on the
left margin represent the molecular weight marker proteins (TEFCO): β-galactosidase (116
kDa), phosphorylase b (97.4 kDa), bovine serum albumin (69,0 kDa), glutamic dehydrogenase
(55.0 kDa), lactic dehydrogenase (36.5 kDa), carbonic anhydrase (29.0 kDa), trypsin inhibitor
(20.1 kDa), lysozyme (14.3 kDa), and aprotinin (6.5 kDa).
Fig. 4. Luminescence reaction of ZZ-gCys, Z-gCys and Z-domain with coelenterazine.
A. Luminescence patterns of ZZ-gCys, Z-gCys and Z-domain (5 μg). Assay conditions are the
same as in Table 2.
B. Luminescence patterns of ZZ-gCys (a:5 μg and b: 15 μg), BSA (5 μg), and ZZ-gCys (5 μg)
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with 0.01% Tween 20. Assay conditions are the same as in Table 2.
C. Luminescence pattern of ZZ-gCys (5 μg) at the presence of a 1:1 molar ratio to IgG (21 μg)
or BSA (19 μg). Assay conditions are the same as in Table 4.
Fig. 5. Luminescence emission spectra of ZZ-gCys, Z-gCys and Z-domain with
coelenterazine.
The measurement conditions are described in Materials and Methods.
Fig. 6. Stimulation of the luminescence activity of ZZ-gCys by Tween 20 (A) and NaCl
(B).
The assay conditions are described in Materials and Methods.
Fig. S1. Effect of pH on the luminescence activity of Z-domain (A), Z-gCys (B) and ZZ-gCys
(C). The reaction mixture (100 μL) contains 5 μg of protein and 1 μg of coelenterazine in
various pH solutions. Closed circle, 100 mM Tris-HCl buffer; Open circle, 100 mM sodium
phosphate buffer; Closed triangle, 100 mM sodium
phosphate buffer.
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