Enhanced IgG1 production by overexpression of nuclear factor kappa B inhibitor zeta (NFKBIZ) in Chinese hamster ovary cells
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Several engineering strategies have been employed to improve the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cell lines. We have focused on unfolded protein response-based engineering and reported that ATF4 overexpression increases protein production. In this study, transcriptome analysis of ATF4-overexpressed CHO cells was performed using high-coverage expression profiling, to search for another key factor contributing to recombinant protein production. We observed the upregulated expression of transcription factor, nuclear factor (NF)-kappa-B inhibitor zeta (NFKBIZ or Iκbζ), in ATF4-overexpressed cells. A total of 1917 bp of CHO NFKBIZ cDNA was cloned, and two stable cell lines overexpressing NFKBIZ were constructed. We investigated the effects of NFKBIZ on IgG1 production in CHO cells. Although the two stable cell lines, NFKBIZ-A and -B, had the opposite phenotypes in cell growth, the specific IgG1 production rate of both cell lines was enhanced by 1.2–1.4-fold. In the NFKBIZ-A cell line, the synergistic effect between enhanced viable cell density and improved specific IgG1 production rate brought about a large increase in the final IgG1 titer. Luciferase-based NF-κB signaling assay results suggest that altered p50/p50 signaling seems to be due to the opposite phenotypes in cell growth. No difference was observed in the translational levels and intracellular assembly states of IgG1 between mock and two NFKBIZ cell lines, indicating that the secretion machinery of correctly folded IgG1 was enhanced in NFKBIZ-overexpressing cell lines.
KeywordsHigh-coverage expression profiling (HiCEP) NF-kappa B inhibitor zeta (NFKBIZ) Antibody production Chinese hamster ovary (CHO) cells
The authors would like to thank Dr. Tomoshi Ohya (Mitsubishi Tanabe Pharma) for his valuable discussions. This research was partially supported by developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses from the Ministry of Economy, Trade and Industry, Japan (METI) and from the Japan Agency for Medical Research and Development (AMED).
- Feger F, Dubart A, Lacout C, Dusanter-Fourt I, Mayeux P, Vainchenker W, Dumenil D (1997) Ectopic expression of the erythropoietin receptor in a murine interleukin-6-dependent plasmacytoma cell line (TEPC-2027) confers proliferative responsiveness to erythropoietin. Blood 89:435–445Google Scholar
- Nelson DE, Ihekwaba AE, Elliott M, Johnson JR, Gibney CA, Foreman BE, Nelson G, See V, Horton CA, Spiller DG, Edwards SW, McDowell HP, Unitt JF, Sullivan E, Grimley R, Benson N, Broomhead D, Kell DB, White MR (2004) Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306:704–708CrossRefGoogle Scholar
- Yamamoto M, Yamazaki S, Uematsu S, Sato S, Hemmi H, Hoshino K, Kaisho T, Kuwata H, Takeuchi O, Takeshige K, Saitoh T, Yamaoka S, Yamamoto N, Yamamoto S, Muta T, Takeda K, Akira S (2004) Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IkappaBzeta. Nature 430:218–222CrossRefGoogle Scholar