Clinical Chemistry 47: 471-476, 2001;
(Clinical Chemistry. 2001;47:471-476.)
© 2001 American Association for Clinical Chemistry, Inc.
Production of Recombinant Human Creatine Kinase (r-hCK) Isozymes by Tandem Repeat Expression of M and B Genes and Characterization of r-hCK-MB
Yoshiko Sunahara1,
Kohji Uchida1,
Toshio Tanaka1,
Hirokazu Matsukawa1,
Manabu Inagaki1 and
Yuhsi Matuoa,1
1
Nagahama Institute for Biochemical Science, Oriental Yeast Co., Ltd., 50 Kano-cho, Nagahama-shi, Shiga 526-0804, Japan.
a Address correspondence to: Fax 81-749-63-7910; e-mail
yushi211{at}oyc.co.jp.
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Abstract
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Background: Serum creatine kinase-MB isoenzyme (CK-MB) is
widely used as a marker of myocardial injury. We prepared recombinant
human CK (r-hCK) MB isoenzyme and examined its potential for use as a
control material for assay of CK-MB in serum.
Methods: cDNAs encoding CK-M and CK-B subunits were
inserted into the same plasmid vector, followed by transformation of
Escherichia coli. The resulting three types of CK
isoenzymes were purified by conventional chromatography.
Results: The ratio of MB to MM to BB was 50:40:10 on the
basis of CK activity. Highly purified CK-MB with a specific activity of
533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified
r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa
for the subunit) of native CK-MB. Its immunoreactivity in an
ELISA using antibody against native heart enzyme was similar to
that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at
least 4 months at 11 °C in a delipidated serum matrix in a liquid
form at a concentration of 118 U/L.
Conclusions: r-hCK-MB shows key properties of the native
cardiac isoenzyme and may be useful as a control and calibrator for
serum assays of CK-MB.
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Introduction
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Assay methods for measuring the activity or protein concentration
of creatine kinase-MB isoenzyme
(CK-MB)1
have been developed as markers of acute myocardial infarction.
Methods for measuring protein concentration (mass assay) required the
establishment of a standard material (1). Many kinds of
controls and calibration materials are used for the various assays. For
controls and calibrators, both stable supply and high quality are
required.
We have applied gene technology to produce 12 kinds of recombinant
proteins, such as human recombinant myoglobin and C-reactive
protein. In this study, we produced recombinant human CK-
(r-hCK)-MB, -MM, and -BB isoenzymes, utilizing recombinant DNA
technology to express heterodimeric isoenzymes.
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Materials and Methods
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materials
Native human CK-MB and r-hCK-MB were purchased from Aalto
Scientific Ltd. and Genzyme Diagnostics, respectively.
cloning of ck-m and ck-b cDNAs
Using human skeletal muscle cDNA library as a template, we used
PCR to amplify cDNA that encodes 1146 bp of the human CK M-type
isoenzyme (2). This amplified DNA was digested with
EcoRI/BamHI and inserted into pBluescript II
SK(-) digested with EcoRI/BamHI to
construct pBluescript II SK(-)-CK-M. The nucleotide sequence of
the cloned DNA was analyzed on an
ALFexpressTM DNA sequencer (Amersham
Pharmacia Biotech UK Ltd.). Expression plasmid pTRP-CK-M was
constructed by insertion of the DNA fragment prepared by digesting the
pBluescript II SK(-)-CK-M with EcoRI/BamHI,
which encodes the human CK M-type subunit, into expression vector pTRP
(3) that had been digested with
EcoRI/BamHI. For expression of 1146 bp of the
human CK B-type isoenzyme (4), plasmid pTRP-CK-B was
constructed according to a method similar to the one mentioned above
using a human brain cDNA library as a template.
construction of ck-mb expression vector
HindIII/SalI-digested pTRP-CK-M fragment was
inserted into pBluescript II SK(-) to construct pBluescript II
SK(-)-trpP+CK-M. This plasmid was digested with XbaI, and
the fragment in which the trp promoter and human CK M-type subunit gene
are located was purified. This DNA fragment was inserted into
XbaI-digested pTRP-CK-B, the 5' terminus of which was
dephosphorylated, to construct pTRP-CK-B/M (Fig. 1
).
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Figure 1. Construction of plasmid for human CK expression.
The genes that encode for the CK-M and -B subunits were cloned from
human cardiac and cerebral cDNA libraries, respectively. The cDNAs
encoding the CK-M and CK-B subunits were inserted as a tandem repeat
expression vector, pTRP (3), and were expressed in E.
coli according to the method described previously
(3).
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This expression vector was transformed into Escherichia
coli. r-hCK-MB, -MM, and -BB were expressed according to the
method described previously (3).
purification of r-hCK-MB
r-hCK-MB was purified from cell lysate by three chromatographic
methods (hydrophobic, ion-exchange, and gel-filtration) after ammonium
sulfate fractionation. The precipitate that formed at 80% saturation
of ammonium sulfate was dissolved in 50 mmol/L sodium phosphate buffer
(pH 8.0) containing 40% saturation of ammonium sulfate, 1
mmol/L MgCl2, 0.2 mmol/L EGTA, and 2 mmol/L
ß-mercaptoethanol, and subjected to hydrophobic chromatography on a
phenyl-Sepharose column with 40–0% linear gradient elution. Fractions
containing CK activity were equilibrated with 50 mmol/L sodium
phosphate buffer (pH 7.4) containing 2 mmol/L ß-mercaptoethanol,
applied to a Q-Sepharose column, and washed with the same buffer.
r-hCK-MB was eluted from the column with a linear NaCl gradient (0–300
mmol/L). r-hCK-MB fractions were subjected to buffer exchange with 20
mmol/L Tris-HCl buffer (pH 8.5) containing 0.15 mol/L NaCl, 10
mmol/L ß-mercaptoethanol, 500 g/L glycerol, and 1 g/L
NaN3 by gel filtration and then stored at
-80 °C. The total protein concentration was determined by the
bicinchoninic acid method (Pierce Chemical Company) (5) with
bovine serum albumin as the calibrator.
The purity of r-hCK-MB was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10–20%
gradient gels (Daiichi Pure Chemicals). Electrophoresis was performed
using Cassette Electrophoresis Unit "DAIICHI", followed by staining
with Rapid Stain Coomassie Brilliant Blue (nacalai tesque).
characterization of r-hCK-MB
The pI of r-hCK-MB was determined on the Phast System (Amersham
Pharmacia Biotech) in a pH range of 3–9. Gels were stained with Phast
Gel Blue R (Coomassie Brilliant Blue R-350).
CK activity was measured at 37 °C according to the method
recommended by the Japanese Society of Clinical Chemistry with the
reaction mixture shown in Table 1
on a Hitachi-7150 automated analyzer unless otherwise stated.
Enzyme samples were diluted up to 
1000 U/L with 20 mmol/L Tris
buffer (pH 8.5) containing 1 g/L bovine serum albumin.
The Km values of r-hCK-MB for creatine
phosphate (CP) and ADP were determined under conditions described in
the Japanese method with CP and ADP at concentrations of 0.1–100 and
0.01–10 mmol/L, respectively. Data were analyzed by Lineweaver-Burk
plots.
To determine optimum pH, enzyme activity was measured using the buffer
shown in Table 1
as a basic solution; 100 mmol/L imidazole or bis-Tris
buffer was used as the buffer for reactions at pH 6.0–7.2.
sandwich elisa for ck-mb
For the ELISA, a 96-well plate was coated with anti-CK-MB
monoclonal antibody (20 g/L), which was cross-reactive with
CK-MB and -BB but not CK-MM, and incubated at 4 °C overnight. The
plate was washed five times with phosphate-buffered saline (PBS)
containing 0.5 g/L Tween 20 (Tween-PBS). PBS containing 10 g/L bovine
serum albumin was added for blocking and incubated for 1 h at room
temperature. After the wells were washed with Tween-PBS, a sample
solution containing CK-MB was added and incubated at room temperature
for 2 h, followed by washing with Tween-PBS. Rabbit anti-CK-BB
polyclonal antibody (10 g/L) was added and incubated at room
temperature for 1 h. After the wells were washed with Tween-PBS,
goat anti-rabbit IgG-horseradish peroxidase conjugate was added
and incubated at room temperature for 1 h, followed by washing
with Tween-PBS. Substrate solutions for horseradish peroxidase,
o-phenylenediamine (0.4 g/L), and
H2O2 were added. After a
10-min incubation at room temperature, the reaction was stopped by the
addition of 12 mol/L
H2SO4. The absorbance at
492 nm was measured using a micro plate reader (MRP A4; TOSOH). Samples
were diluted to final concentrations of 0, 12.5, 25, 50, 100, and 200
µg/L, and the absorbances measured at these concentrations were
plotted.
immunoinhibition assay
r-hCK-MB, -MM, and -BB samples were diluted to a CK activity of
2500 kU/L in 9 g/L NaCl containing 70 g/L human serum
albumin. Goat anti-CK-MM antibody (40.1 g/L), an inhibitor of
CK-M subunit activity, was diluted with the NaCl-human serum albumin
solution to prepare five different concentrations. The NaCl-human serum
albumin solution was used as the zero inhibitor concentration. Antigen
solution (50 µL) was added per 200 µL of sample, followed
by incubation at room temperature for 10 min. The remaining CK activity
was measured using the Merck assay for CK activity (Kanto
Chemical Co.) on a Hitachi-7150 automated analyzer (Hitachi). The
remaining activity at each dilution was calculated by defining the zero
antigen concentration as 100%.
stability of r-hCK-MB
To study the stability of r-hCK-MB in buffer, r-hCK-MB was diluted
to 2000 kU/L in 20 mmol/L Tris-HCl buffer (pH 8.5) containing 0.15
mol/L NaCl, 10 mmol/L ß-mercaptoethanol, 500 g/L glycerol, and 1 g/L
NaN3 and stored at -80 or 4 °C. To study the
stability of r-hCK-MB in a serum matrix, r-hCK-MB was diluted to 120
U/L in delipidated serum purchased from Strategic BioSolutions and
stored at -80 or 11 °C. r-hCK-MB stability was examined by an
immunoinhibition assay (6) using the CK-MB E-HA Test (Wako
Pure Chemical Industries).
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Results and Discussion
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expression and purification
The insertion of cDNAs that encoded for CK-M and CK-B subunits
into the same plasmid vector enabled three isoenzymes, r-hCK-MB, -MM,
and -BB, to be expressed simultaneously in E. coli.
r-hCK-MB was purified from crude extract by ammonium sulfate
fractionation, followed by hydrophobic, anion-exchange, and
gel-filtration chromatography (Table 2
). Three CK peaks were eluted from the anion-exchange
column (Fig. 2
). The first peak did not react with the anti-CK-MB antibody.
The second and third peaks were identified as CK-MB and CK-BB,
respectively, based on their cross-reactivity to the antibody, and were
confirmed by pI. r-hCK-MM was eluted in the flow-through fraction,
whereas the other two isoenzymes were bound to the column, and r-hCK-MB
was eluted at 125 mmol/L NaCl. r-hCK-MB with a specific activity of
533 U/mg was obtained in a yield of 12%. The isoelectric points of
these three recombinant CK enzymes were indistinguishable from those of
their native counterparts, suggesting that the native proteins are
minimally processed. The expression ratio of these three types of
isoenzymes was MM:MB:BB = 40:50:10 on the basis of enzyme
activity. Thus, the technology allowed production of all three types of
CK.
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Figure 2. Purification of r-hCK-MB by Q-Sepharose anion-exchange
chromatography.
Eluates from phenyl-Sepharose CL-4B were subjected to ultrafiltration
to change the buffer to 50 mmol/L sodium phosphate (pH 8.5) containing
2 mmol/L ß-mercaptoethanol and then were applied to a 50-mL column.
r-hCK-MB was eluted with a linear gradient of NaCl (solid
line) from 0 to 0.3 mol/L.
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purity of r-hCK-MB
The estimated purity of r-hCK-MB was 90% when 2 µg of
protein was analyzed by SDS-PAGE. The molecular mass of the
subunit was 46 kDa (Fig. 3
), and the isoelectric point was 5.2 (data not shown),
consistent with the reports of Kanemitsu and Okigaki
(7)(8). No band was detected at pI 6.5, which
corresponds to CK-MM, or pI 4.5, which corresponds to CK-BB, even when
10 µg of enzyme protein was applied, suggesting that the
contamination of r-hCK-MB with r-hCK-MM or -BB was <1%
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Figure 3. SDS-PAGE analysis.
Lane M, molecular mass marker [pA(AB)1–6;
Oriental Yeast Co Ltd.]; lane 1, E. coli
cell lysate (20 µg of protein); lane 2, 40% ammonium
sulfate supernatant (20 µg of protein); lane 3, 80%
ammonium sulfate precipitate (20 µg of protein); lane
4, phenyl-Sepharose CL-4B eluate (10 µg of protein);
lane 5, Q-Sepharose eluate (2 µg of protein);
lane 6, Sephacryl S-200 (final preparation; 2 µg of
protein). Each sample was subjected to SDS-PAGE as described in
Materials and Methods.
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properties
The enzymologic properties of purified r-hCK-MB were examined by
comparing with native hCK-MB or the r-hCK-MB from Genzyme, which has
been characterized as a reference material. The
Km values were 1.2 x
10-3 mol/L for CP and 1.3 x
10-4 mol/L for ADP. The activity was maximum at
pH 6.2–6.8 in imidazole buffer as well as in bis-Tris buffer (Table 3
). The immunologic reactivity of r-hCK-MB was evaluated by
comparing it to native hCK-MB and the r-hCK-MB from Genzyme, as assayed
by sandwich ELISA (Fig. 4
). The ELISA results, using anti-CK-MB monoclonal antibody as
capture antibody and anti-CK-BB antibody as the detection antibody,
indicated that the response curve for r-hCK-MB was similar to that of
native CK-MB. The immunologic reactivities of r-hCK-MB, -MM, -BB;
native hCK-MB; and the r-hCK-MB from Genzyme were analyzed by
immunoinhibition assay using an anti-CK-MM antibody that specifically
inhibits M-type subunit activity (Fig. 5
). The r-hCK-MB activity remaining at a concentration of
anti-CK-MM antibody at which M-type activity was entirely inhibited was
42.4%, which was similar to the reactivity of native hCK-MB (42.8%)
and the Genzyme r-hCK-MB (43.1%), whereas the remaining activity for
r-hCK-BB was 99.4%. These results indicate that r-hCK-MB possesses
immunoreactivity similar to that of the native enzyme.
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Figure 4. Immunoreactivity of r-hCK-MB.
Samples were assayed by sandwich ELISA using anti-CK-MB monoclonal
antibody as capture antibody and anti-CK-BB antibody as detection
antibody.
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Figure 5. Immunoinhibition of r-hCK-MB.
r-hCK-MB, -MM, and -BB samples were incubated for 10 min at room
temperature with five 1:2 serial dilutions of goat anti-CK-MM antibody,
which inhibits CK-M subunit activity. The remaining CK activity was
measured using Merckliquid CK (Kanto), the reagent set for
measuring CK activity, and a Hitachi-7150 automated analyzer.
The remaining activity of each sample was calculated by defining the
value of sample containing no anti-CK-MM antibody as 100%.
 , native hCK-MM (Aalto); •, r-hCK-MM from this study;  ,
native hCK-MB (Aalto);  , r-hCK-MB (Genzyme);  , r-hCK-MB from this
study;  , r-hCK-BB from this study.
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stability
In a buffer matrix, r-hCK-MB activity was stable for 20 days at
-80 °C, whereas at 4 °C, a 15% loss of activity was observed
after 15 days (Fig. 6
). To study stability in a serum matrix, buffer matrix
containing r-hCK-MB was diluted 1:100 with 9 g/L NaCl, and further
diluted with delipidated serum to an activity of 120 U/L. It was
stable at least for 9 months at -80 °C. At 11 °C, 93.2% of the
original activity remained after 4 months, and 75% remained after 9
months (Fig. 7
).
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Figure 6. Stability of r-hCK-MB in buffer matrix.
r-hCK-MB was dissolved with 20 mmol/L Tris-HCl (pH 8.5) containing 0.15
mol/L NaCl, 10 mmol/L ß-mercaptoethanol, 500 g/L glycerol, and 1
g/L NaN3 and stored at -80 or 4 °C. At the
respective temperatures, the day 0 value was defined as 100%.
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Figure 7. Stability of r-hCK-MB in serum matrix.
r-hCK-MB was formulated in delipidated normal serum at a concentration
of 120 U/L and stored at -80 or 11 °C.
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In conclusion, the results indicate that r-hCK-MB has the potential
to be useful as a control/calibrator.
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Footnotes
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1 Nonstandard abbreviations: CK, creatine kinase; r-hCK-MB, -MM, and -BB, recombinant human creatine kinase-MB, MM, and BB isozymes; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CP, creatine phosphate; and PBS, phosphate-buffered saline. 
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References
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Abstract
Introduction
