Understanding the enhanced effect of dimethyl sulfoxide on hepatitis B surface antigen expression in the culture of Chinese hamster ovary cells on the basis of proteome analysis

doi:10.1016/j.enzmictec.2005.05.016

Enzyme and Microbial Technology

Volume 38, Issues 3–4, 1 February 2006, Pages 372-380

https://doi.org/10.1016/j.enzmictec.2005.05.016 Get rights and content

Abstract

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 HBsAg in cells. The post-translational modification and secretion of HBsAg potentially became the limiting-step in further improvement of protein expression with DMSO.

Introduction

The Chinese hamster ovary (CHO) cell line is an excellent host for producing complex recombinant proteins. It is able to synthesize and secrete recombinant glycoproteins with carbohydrate structures similar to those found in humans. This is an important consideration in biopharmaceutical glycoprotein production since the carbohydrate moiety plays a critical role in biological activity and plasma clearance rate [1]. Therefore, recombinant proteins produced by mammalian cells are mostly superior to those produced by bacteria or yeast. However, the productivity of mammalian cell expression systems is much lower than that of microorganism systems [2]. Many strategies are being used to improve the productivity, such as the selection of preferable codons, the supplementation of expression stimulatory agents, metabolic regulation and bioprocessing optimization [3], [4], [5], [6].

Among them, the supplementation of stimulatory chemical agents is an effective, convenient and quick method. The specific secretion rate of recombinant erythropoietin (EPO) was increased by almost 8-folds in presence of 5 mM cysteasmine [7]. Butyrate, another stimulatory agent used for extra-protein expression [8], has been used to increase the production of tissue plasminogen activator (tPA), epidermal growth factor (EGF) receptor and human gonadotropin proteins in mammalian cell cultures. Balcarcel and Stephanopoulos [9] reported that rapamycin enhanced specific monoclonal antibody production with little effect on cell growth. Dimethyl sulfoxide (DMSO), a novel candidate for protein synthesis enhancer in mammalian cell culture, improved specific production rates of monoclonal antibody in hybridoma cells by 2-fold and a fusion protein in CHO cells by 1.6-fold [10], [11].

Nevertheless, the mechanism of DMSO effects on protein expression and cell metabolism remains unclear. To our knowledge, the response of recombinant CHO cells to DMSO has not been reported so far. The proteomic approach has been considered as a good way to characterize global alterations in protein expression of cells. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are the key techniques in proteomic research. The combination of physical protein data from mass spectrometry with genome data enables unambiguous identification of proteins from 2-DE gels more rapidly and cheaply. Coupled with protein identification technology and associated database search engines, a proteomic approach can be feasibly applied to investigate the cytophysiological changes. In mammalian cell culture for recombinant protein production, investigation of intracellular response to stimulatory chemical agents and environmental factors will do a good job to direct our further efforts on process optimization. Lee et al. [12] had found a few significant proteins in CHO cells in response to hyperosmotic pressure by proteomic analysis, such as pyruvate kinase, glyceraldehyde 3-phosphate dehydrogenase and tubulin. In the study of the response of CHO cells to butyrate and zinc sulphate, Dyk et al. [13] found that the chaperone proteins, GRP78, HSP90, GRP94 and HSP70, were not up-regulated by proteomic approach. Kaufmann et al. [14] found that CHO cells synthesized specific cold-inducible proteins in response to a low cultivation temperature shift by two-dimensional gel electrophoresis. These works provide good clues to understand the cellular response to culture conditions.

In this work, the proteomic approach was applied to investigate the intracellular protein expression profiles of recombinant CHO cells in response to DMSO. It aims to improve the understanding of recombinant product processing in CHO cells and the mechanism of DMSO effects.

Section snippets

Cell line and medium

The cell line CHO-C₂₈, genetically modified to express recombinant hepatitis B surface antigen (HBsAg), was kindly provided by Wuhan Biologicals Institute (Wuhan, China). The cell line was maintained in 25 cm² T-flasks under 5% CO₂ at 37 °C, subcultured every 3 or 4 days by trypsinization and dilution with Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% (v/v) newborn bovine serum (NBS, Sijiqing Biotechnology Co., Hangzhou, China). The basal medium used in all cultures was

Cell growth and HBsAg production in response to DMSO

In the pulse culture with DMSO, DMSO could be clearly seem to depress CHO cell growth above 27% in presence of 0.5–2.5% DMSO (Fig. 1A). The viable cell density decreased by about 80% when the DMSO concentration was increased to 2.0%. However, the cell viability is still kept above 85% in spite of the DMSO addition in 0–2.5%. The cell death and lysis were not apparent at least in pulse culture.

DMSO enhanced both the production and productivity of HBsAg (Fig. 1B). HBsAg production and

Discussion

CHO cells have been widely used to produce recombinant proteins for therapeutic and diagnostic purposes. However, the low productivity of this expression system limited its application. DMSO and pentanoic acid have been demonstrated their ability to enhance the protein production [6], [10], [11]. However, the molecular mechanism of their effects is largely unknown. To better understand intracellular responses of recombinant CHO cells to DMSO, we applied two-dimensional (2-D) gel electrophoresis

Acknowledgements

This work was partially funded by the project of Chinese National Programs for High Technology Research and Development (2002AA217011) and the grant of Shanghai Key Discipline of Biochemical Engineering.

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The intracellular hepatitis B surface antigen (HBsAg) content per cell was significantly increased by 7.2-fold in the culture of recombinant CHO cells with 1.5% dimethyl sulfoxide (DMSO), while the HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. The significant accumulation of HBsAg within rCHO cells by DMSO stimulation was testified with flow cytometry measurements. Electron microscopy was applied to show that the dilated areas scattered over whole cytoplasm within rCHO cells in response to DMSO, and further revealed that intracellular HBsAg was localized to these areas with immunogold labeling. The failure of intracellular HBsAg virus-like particle assembly was revealed to be closely associated with the HBsAg accumulation within DMSO-stimulated rCHO cells on the basis of sucrose gradient analysis of cell extract. This work provided the details to further understand the HBsAg accumulation within rCHO cells in response to DMSO.

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Understanding the enhanced effect of dimethyl sulfoxide on hepatitis B surface antigen expression in the culture of Chinese hamster ovary cells on the basis of proteome analysis

Abstract

Introduction

Section snippets

Cell line and medium

Cell growth and HBsAg production in response to DMSO

Discussion

Acknowledgements

Gene

J Biosci Bioeng

J Neurosci Methods

Anal Biochem

Protein Express Purif

Trends Cell Biol

FEBS Lett

FEBS Lett

Glycosylation of recombinant proteins: problems and prospects

Enzyme Microbiol Technol

Overview of vector design for mammalian gene expression