Proteomic Profiling of Temperature-Shifted CHO Cells to Identify Genes that Impact on Both Cell Growth and Recombinant Protein Productivity in Suspension Culture

Numerous cellular responses occur when cultured cells are shifted from growth at 37 degrees C to lower temperatures. This method known as temperature shift (TS) is frequently employed during recombinant protein (rP) production processes to restrict further biomass accumulation and boost rP productivity. In this study we used a 2D-DIGE/MALDI-TOF approach to identify genes whose expression in suspension-adapted CHO cells was altered as a result of the shift from growth at 37 to 31 degrees C. 23 proteins were found to be differentially expressed subsequent to the TS and were identified by mass spectrometry. One of these, Eukaryotic translation initiation factor 4a isoform 1 (eiF4A), was chosen for functional validation studies to decipher its role in the cellular response to hypothermic conditions. Part of the gene transcript was amplified by PCR using primers based on comparison of orthologous sequences in order to generate enough sequence for siRNA design. eiF4a expression was altered in small-scale suspension cultures of SEAP-secreting CHO cells. Cultures were assessed for cell growth and rP productivity after transient transfection with siRNA. When the expression of the translation factor was knocked down, it was found to reduce growth of cells in both TS and constant temperature (37 degrees C) cultures and to improve productivity in TS cultures. In conclusion, we present an approach to identify potentially useful gene engineering targets via proteomic expression analysis of temperature shifted CHO cells and subsequent validation by RNA interference (RNAi).