Journal of Biotechnology 146 (2010) 160–168 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system Teng Rhui Lwa a , Chuan Hao Tan a , Qiao Jing Lew a , Kai Ling Chu a , Janice Tan b , Yih Yean Lee b , Sheng-Hao Chao a,∗ a Expression Engineering Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668, Singapore b Animal Cell Technology Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Singapore 138668, Singapore article info Article history: Received 25 September 2009 Received in revised form 8 January 2010 Accepted 18 February 2010 Keywords: CEBPG CHO HEK293 EPO Monoclonal antibody siRNA 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 bio- pharmaceutical 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 tran- siently 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 contain- ing 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 recombi- nant EPO, interferon , 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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Most of biopharmaceutical proteins, such as erythropoietin (EPO), interferon, and therapeutic monoclonal antibodies (mAbs), are secreted proteins. Production of recombinant secreted pro- teins in mammalian cells is tightly regulated by several biological processes, including transcription, post-transcriptional processing, translation, post-translational modification, protein folding, and secretion. It is very challenging and time-consuming to identify a specific gene from one of these processes to further confirm its involvement in the production of recombinant biopharmaceutical proteins in mammalian cells, such as human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO) cells. Recently, functional genomic high-throughput screens, including cDNA and siRNA screens, have been utilized for quick target identification of specific biological processes and pathways (Huang et al., 2004; Aza- Blanc et al., 2003). This screening system, of course, can be applied to isolate the cellular target genes required for production of secreted proteins, including biopharmaceutical proteins. If the screening assays are appropriately designed, the screen can specifically pick up the crucial cellular targets from any of ∗ Corresponding author. Tel.: +65 6407 0899; fax: +65 6478 9561. E-mail address: jimmy chao@bti.a-star.edu.sg (S.-H. Chao). the biological pathways that regulate productivity of secreted proteins. Luciferase is the popular choice of the reporter protein used in cell-based screens for quick detection. Firefly and Renilla luciferases (Fluc and Rluc) are two most commonly used luciferases in these assays. However, both luciferases are not secreted proteins and remain inside the cell after being synthesized (Tannous et al., 2005). Therefore, an increase in Fluc and Rluc does not neces- sarily guarantee the increased yield of secreted proteins in the culture media. To identify the critical genes required for the produc- tion of secreted proteins, a secreted luciferase, Gaussia luciferase (Gluc) (Tannous et al., 2005), could be a better choice for this purpose. Human cytomegalovirus (CMV) major immediate-early (MIE) promoter, one of the most potent RNA polymerase II promoters, is widely used in HEK293 and CHO cells for production of recombi- nant proteins. In this study, we generated a Gluc reporter plasmid, pCMV-Gluc (in which the expression of Gluc was driven by the CMV MIE promoter) and used it to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potas- sium channel tetramerisation domain containing 2 (KCTD2), and transmembrane protein 183A (TMEM183A), were isolated from the siRNA Gluc screen. Knockdown of CEBPG, KCTD2, and TMEM183A resulted in significant decreases EPO production, confirming the practical use of this novel Gluc-based siRNA screening system for 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.02.016