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RISS 인기검색어
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- 저자 : Yusufi, F.N.K. (검색결과 3 건)
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Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line
Yusufi, F.N.K.,Lakshmanan, M.,Ho, Y.S.,Loo, B.L.W.,Ariyaratne, P.,Yang, Y.,Ng, S.K.,Tan, T.R.M.,Yeo, H.C.,Lim, H.L.,Ng, S.W.,Hiu, A.P.,Chow, C.P.,Wan, C.,Chen, S.,Teo, G.,Song, G.,Chin, J.X.,Ruan, X. Cell Press 2017 Cell systems Vol.4 No.5
Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner.
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Enhanced expression of codon optimized interferon gamma in CHO cells
Chung, B.K.S.,Yusufi, F.N.K.,Mariati,Yang, Y.,Lee, D.Y. Elsevier Science Publishers 2013 Journal of biotechnology Vol.167 No.3
The human interferon-gamma (IFN-γ) is a potential drug candidate for treating various diseases due to its immunomodulatory properties. The efficient production of this protein can be achieved through a popular industrial host, Chinese hamster ovary (CHO) cells. However, recombinant expression of foreign proteins is typically suboptimal possibly due to the usage of non-native codon patterns within the coding sequence. Therefore, we demonstrated the application of a recently developed codon optimization approach to design synthetic IFN-γ coding sequences for enhanced heterologous expression in CHO cells. For codon optimization, earlier studies suggested to establish the target usage distribution pattern in terms of selected design parameters such as individual codon usage (ICU) and codon context (CC), mainly based on the host's highly expressed genes. However, our RNA-Seq based transcriptome profiling indicated that the ICU and CC distribution patterns of different gene expression classes in CHO cell are relatively similar, unlike other microbial expression hosts, Escherichia coli and Saccharomyces cerevisiae. This finding was further corroborated through the in vivo expression of various ICU and CC optimized IFN-γ in CHO cells. Interestingly, the CC-optimized genes exhibited at least 13-fold increase in expression level compared to the wild-type IFN-γ while a maximum of 10-fold increase was observed for the ICU-optimized genes. Although design criteria based on individual codons, such as ICU, have been widely used for gene optimization, our experimental results suggested that codon context is relatively more effective parameter for improving recombinant IFN-γ expression in CHO cells.
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Combined in silico modeling and metabolomics analysis to characterize fed‐batch CHO cell culture
Selvarasu, Suresh,Ho, Ying Swan,Chong, William P. K.,Wong, Niki S. C.,Yusufi, Faraaz N. K.,Lee, Yih Yean,Yap, Miranda G. S.,Lee, Dong‐,Yup Wiley Subscription Services, Inc., A Wiley Company 2012 Biotechnology and bioengineering Vol.109 No.6
<P><B>Abstract</B></P><P>The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency and yield of these biopharmaceutical products from mammalian cells, which is fully achievable only through proper understanding of cellular functioning. Towards this end, the current study exploited a combined metabolomics and in silico modeling approach to gain a deeper insight into the cellular mechanisms of Chinese hamster ovary (CHO) fed‐batch cultures. Initially, extracellular and intracellular metabolite profiling analysis shortlisted key metabolites associated with cell growth limitation within the energy, glutathione, and glycerophospholipid pathways that have distinct changes at the exponential‐stationary transition phase of the cultures. In addition, biomass compositional analysis newly revealed different amino acid content in the CHO cells from other mammalian cells, indicating the significance of accurate protein composition data in metabolite balancing across required nutrient assimilation, metabolic utilization, and cell growth. Subsequent in silico modeling of CHO cells characterized internal metabolic behaviors attaining physiological changes during growth and non‐growth phases, thereby allowing us to explore relevant pathways to growth limitation and identify major growth‐limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth‐related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance. Biotechnol. Bioeng. 2012; 109:1415–1429. © 2012 Wiley Periodicals, Inc.</P>
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