Improvement of protein production in recombinant CHO cell by proteomic approaches

Abstract

Chinese hamster ovary (CHO) cells have been popular hosts for the commercial production of therapeutic proteins including erythropoietin, antibodies, and antibody-based proteins. For the purpose of higher protein production, numerous omics-based approaches such as genomics, proteomics, transcriptomics, and metabolomics have been tried to understand intracellular events in mammalian cells. Among them, two-dimensional gel electrophoresis (2DE) combined with mass spectrometric (MS) analysis is one of the widely used tools for identifying intracellular factor changes under various culture conditions or interacts with desired therapeutic protein in mammalian cells. Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahistidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by 2DE and identified by MALDI-TOF MS/MS. A total of 27 protein spots including glucose regulated protein 78 (GRP78) were successfully identified. 2D-Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. The glycosylation state in glycoproteins including EPO is important in biosynthesis, secretion, and biological activity. In order to find the intracellular molecules differentially bound with glycosylated or non-glycosylated EPO, fully glycosylated EPO having three N-linked glycosylation sites was purified from EPO-producing rCHO cells by sequential chromatography using heparin c...