Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB.
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 degrees 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.
Sunahara
Y
Y
Nagahama Institute for Biochemical Science, Oriental Yeast Co., Ltd., 50 Kano-cho, Nagahama-shi, Shiga 526-0804, Japan.
Uchida
K
K
Tanaka
T
T
Matsukawa
H
H
Inagaki
M
M
Matuo
Y
Y
D003402:Creatine Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000592:standards; D052276:Creatine Kinase, BB Form; D052279:Creatine Kinase, MB Form; D052283:Creatine Kinase, MM Form; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000592:standards; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards
D018076:DNA, Complementary; D004274:DNA, Recombinant; D007527:Isoenzymes; D011994:Recombinant Proteins; D003402:Creatine Kinase; D052276:Creatine Kinase, BB Form; D052279:Creatine Kinase, MB Form; D052283:Creatine Kinase, MM Form