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 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.
Lwa
Teng Rhui
TR
Expression Engineering Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668, Singapore.
Tan
Chuan Hao
CH
Lew
Qiao Jing
QJ
Chu
Kai Ling
KL
Tan
Janice
J
Lee
Yih Yean
YY
Chao
Sheng-Hao
SH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D048708:Cell Growth Processes / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D022762:CCAAT-Enhancer-Binding Proteins; C405462:CCAAT-enhancer-binding protein-gamma; D008565:Membrane Proteins; D024642:Potassium Channels, Voltage-Gated; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C513550:TMEM183A protein, human; D004921:Erythropoietin; D007371:Interferon-gamma; D008156:Luciferases