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Production of native creatine kinase B in insect cells using a baculovirus expression vector

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Abstract

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

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Authors and Affiliations

  1. Department of Cell Biology and Histology, Medical Faculty University Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands

    Yvette J. M. de Kok, Monique P. A. Geurds, Erik A. Sistermans & Bé Wieringa

  2. Department of Virology, Agricultural University, Wageningen, The Netherlands

    Magda Usmany & Just M. Vlak

Authors
  1. Yvette J. M. de Kok
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  2. Monique P. A. Geurds
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  3. Erik A. Sistermans
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  4. Magda Usmany
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  5. Just M. Vlak
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  6. Bé Wieringa
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de Kok, Y.J.M., Geurds, M.P.A., Sistermans, E.A. et al. Production of native creatine kinase B in insect cells using a baculovirus expression vector. Mol Cell Biochem 143, 59–65 (1995). https://doi.org/10.1007/BF00925927

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  • DOI: https://doi.org/10.1007/BF00925927

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