title: 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.
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.
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, People's Republic of China.
authors.forename: Yaohua
authors.initials: Y
authors.lastname: Zhong
authors.forename: Xuan
authors.initials: X
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Xiao
authors.forename: Songhuan
authors.initials: S
authors.lastname: Wei
authors.forename: Tianhong
authors.initials: T
authors.lastname: Wang
mesh_terms: D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007218:Industrial Microbiology; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D021382:Protein Sorting Signals; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004921:Erythropoietin; D043366:Cellulose 1,4-beta-Cellobiosidase