The productivity of hepatitis B surface antigen (HBsAg) in the culture of recombinant Chinese hamster ovary (CHO) cells was enhanced 5-fold more at 1.5% (v/v) dimethyl sulfoxide (DMSO) than that of the control culture without DMSO, while a large quantity of HBsAg was observed inside cells. In order to disclose the molecular mechanism behind these phenomena, a proteomic approach together with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was applied to identify the proteins related to the cellular response to the DMSO presence. Four enzymes related to glycolysis, such as aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and phosphoglycerate kinase, were identified to be down-regulated in the presence of DMSO. Their low activities might correspond to the redistribution of substrate metabolism flux to the enhanced HBsAg expression. On the other hand, HSP70 and ERP57, two important chaperone proteins which play an important role in protein post-translational modification and secretion, were not up-regulated by DMSO, and the unmatched protein processing with the increase of protein expression could result in the accumulation of H13sAg in cells. The post-translational modification and secretion of H13sAg potentially became the limiting-step in further improvement of protein expression with DMSO. (c) 2005 Elsevier Inc. All rights reserved.