Clinical Chemistry
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Clinical Chemistry 47: 471-476, 2001;
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(Clinical Chemistry. 2001;47:471-476.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

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.

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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[Abstract] [Full Text] [PDF]


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