Human creatine kinase MB (hCKMB) is one of the most preferred biomarkers used for the diagnosis of acute coronary syndrome due to its high sensitivity and specificity. The increasing need for highly purified and biologically active hCKMB in the field of diagnostics makes its production valuable. Currently, the production of hCKMB is mainly achieved in methylotrophic yeast, Pichia pastoris, because the production in Escherichia coil is challenging and generally yields an inactive enzyme with a low quantity. With the aim of finding the best way for the high-yield production of active hCKMB in E. coil, an efficient strategy was developed using a construct allowing tandem expression of each subunit with 2 different tags. The strategy allowed the efficient expression and separate characterization of each subunit and 1-step purification of the heterodimeric protein into homogeneity. The heterodimeric protein displayed more than 11-fold greater specific activity than the commercially available one. The production strategy described in this study shows a clear advantage over the currently used ones and can be made available not only for laboratory scale production but also for commercial production. Our study is also a well-suited example for the studies in which novel protein expression strategies are needed to achieve greater yields with higher purities. (C) 2018 S. Karger AG, Basel.