Effect of doxycycline-regulated erp57 expression on thrombopoietin productivity in recombinant chinese hamster ovary cells = 독시사이클린으로 조절되는 ERp57 발현이 재조합 CHO 세포에서 트롬보포이틴의 생산성에 미치는 영향

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In an attempt to increase the specific thrombopoietin (TPO) productivity $(q_TPO)$ of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), 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 doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by co-transfection 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. The doxycycline concentration of 1㎍/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 due to 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.
Advisors
Lee, Gyun-Minresearcher이균민researcher
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2002
Identifier
177024/325007 / 020013655
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 2002.8, [ vi, 46 p ]

Keywords

Doxycycline; ERp57; Thrombopoietin; Tet-Off System; 독시사이클린; ERp57; 트롬보포이틴; Chinese hamster ovary cells

URI
http://hdl.handle.net/10203/27976
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=177024&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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