Effect of doxycycline-regulated protein disulfide isomerase expression on the specific productivity of recombinant CHO cells: thrombopoietin and antibody.
Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. This study attempts to investigate the effect of PDI expression level on specific productivity (q) of recombinant Chinese hamster ovary (rCHO) cells producing thrombopoietin (TPO) and antibody (Ab). To regulate the PDI expression level, the Tet-Off system was introduced in TPO and Ab producing CHO cells, and stable Tet-Off cells (TPO-Tet-Off and Ab-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (Tet-TPO-PDI and Tet-Ab-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and Ab-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of the secreted TPO and antibody. We cultured two Tet-TPO-PDI and two Tet-Ab-PDI clones, and all these clones showed an average of 2.5-fold increase in PDI expression when compared to the basal level. In both these cell lines the PDI expression was tightly controlled by various concentrations of doxycycline. The q of TPO (q(TPO)) was unaffected but that of antibody producing cells was increased by 15-27% due to the PDI expression level.
Mohan
Chaya
C
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Park
Soon Hye
SH
Chung
Joo Young
JY
Lee
Gyun Min
GM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000008:administration & dosage*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013926:Thrombopoietin; D019704:Protein Disulfide-Isomerases; D004318:Doxycycline