Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system

Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since...

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Published inJournal of biotechnology Vol. 146; no. 4; pp. 160 - 168
Main Authors Lwa, Teng Rhui, Tan, Chuan Hao, Lew, Qiao Jing, Chu, Kai Ling, Tan, Janice, Lee, Yih Yean, Chao, Sheng-Hao
Format Journal Article
LanguageEnglish
Published Amsterdam Elsevier B.V 15.04.2010
Elsevier
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Abstract Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon γ, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.
AbstractList Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon γ, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.
Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon I[sup3, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.
Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.
Author Tan, Chuan Hao
Chu, Kai Ling
Lwa, Teng Rhui
Lew, Qiao Jing
Tan, Janice
Lee, Yih Yean
Chao, Sheng-Hao
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Issue 4
Keywords HEK293
siRNA
CHO
Monoclonal antibody
EPO
CEBPG
RNA interference
Enzyme
Luciferase
Identification
CHO cell line
Gene silencing
Screening
Gene
Production
Oxidoreductases
Recombinant protein
Language English
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(c) 2010 Elsevier B.V. All rights reserved.
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Snippet Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors...
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SubjectTerms Animals
Antibodies, Monoclonal - genetics
Antibodies, Monoclonal - metabolism
Biological and medical sciences
Biotechnology
CCAAT-Enhancer-Binding Proteins - genetics
CCAAT-Enhancer-Binding Proteins - metabolism
CEBPG
Cell Growth Processes - physiology
Cell Line
CHO
CHO Cells
Cricetinae
Cricetulus
EPO
Erythropoietin - genetics
Erythropoietin - metabolism
Fundamental and applied biological sciences. Psychology
Gaussia
HEK293
Humans
Interferon-gamma - genetics
Interferon-gamma - metabolism
Luciferases - genetics
Luciferases - metabolism
Membrane Proteins - genetics
Membrane Proteins - metabolism
Monoclonal antibody
Potassium Channels, Voltage-Gated - genetics
Potassium Channels, Voltage-Gated - metabolism
Protein Engineering - methods
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
RNA, Small Interfering - genetics
siRNA
Title Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system
URI https://dx.doi.org/10.1016/j.jbiotec.2010.02.016
https://www.ncbi.nlm.nih.gov/pubmed/20188772
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