title: Effect of doxycycline-regulated ERp57 expression on specific thrombopoietin productivity of recombinant CHO cells.
abstract: In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), the effect of expression level of ERp57, an isoform of protein disulfide isomerase, on q(TPO) was investigated. To regulate ERp57 expression level, the Tet-Off system was first introduced in TPO-33 cells and stable Tet-Off cells (TPO-33-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by cotransfection of pTRE-ERp57 and pTK-Hyg expression vectors into TPO-33-Tet-Off cells and subsequent screening by Western blot analysis of ERp57 and an enzyme-linked immunosorbent assay of secreted TPO. Western blot analysis showed that ERp57 expression level in TPO-33-ERp57 cells could be regulated tightly by the addition of different concentrations of doxycycline to a culture medium. A doxycycline concentration of 1 microg/mL, which did not influence cell growth and TPO production of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expression to a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expression level was obtained in the absence of doxycycline. This increased expression level of ERp57 resulted in a 2.1-fold increase in q(TPO) without growth inhibition, probably as a result of the chaperone-like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that q(TPO) of rCHO cells can be increased by elevating the expression level of ERp57.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea.
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Hwang
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Chung
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism*; D007535:Isomerases / Q000187:drug effects / Q000378:metabolism*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003470:Culture Media; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D013926:Thrombopoietin; D007535:Isomerases; D004318:Doxycycline