Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum-free suspension culture

Abstract

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum-free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum-free media (SFM4CHO (TM) and SF-L1), proteome analyses were carried out using 2- D PAGE and based on spot intensities, 58 high-intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI-TOF-MS, and MS/MS. Compared with the results in serum-containing medium, six proteins, four de novo synthesis of nucleotides-related proteins (dihydrolipoamide S-acetyltransferase, transaldolase, inosine-5'-monophosphate dehydrogenase 2, and lymphoid-restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO (TM). From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 1015% enhanced cell concentration during serum-free adaptation and 1533% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum-free suspension culture. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011