Abstract
The human erythropoietin (HuEPO) structural gene was fused with the secretion signal of the Trichoderma reesei cellobiohydrolase I and controlled by a newly optimized cbh1 promoter in an integrated expression vector pTrCBH-EPO. The recombinant HuEPO construct was transformed into two different T. reesei strains, a protease-deficient strain RutC-30 M3, and a glycosylation-modified strain T108. After lactose induction, the heterologous rHuEPO was found to be stably expressed in the selected transformants T47 (derived from RutC-30 M3) and T112 (derived from T108), which were shown to have high genetic stability. Secretion of erythropoietin in these transformants was further confirmed by SDS-PAGE and western blot analysis. Moreover, the secreted rHuEPO from T112 had an apparent molecular weight of 32 kDa, which was higher than from T47 (28 kDa) and similar to that of mammals (more than 30 kDa). These results demonstrate the potential of using industrial filamentous fungi for the production of human-derived erythropoietin.
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Acknowledgments
The authors acknowledge supports for the fulfillment of this work by the National Natural Science Foundation of China (No. 30800024; No. 30970026) and National Basic Research Program of China (No.2011CB707403). We thank Qingsheng Qi for kindly providing the plasmid pGEM-T-EPO.
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Yaohua Zhong and Xuan Liu contributed equivalently to the work.
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Zhong, Y., Liu, X., **ao, P. et al. Expression and Secretion of the Human Erythropoietin Using an Optimized cbh1 Promoter and the Native CBH I Signal Sequence in the Industrial Fungus Trichoderma reesei . Appl Biochem Biotechnol 165, 1169–1177 (2011). https://doi.org/10.1007/s12010-011-9334-8
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DOI: https://doi.org/10.1007/s12010-011-9334-8