서지주요정보
Foreign protein production and cellular protein expression in recombinant CHO cells subjected to osmotic and low culture temperature stress = 삼투압 및 저온 배양 stress에 의한 재조합 CHO 세포의 외래단백질 생산 및 세포 내 단백질 발현 변화
서명 / 저자 Foreign protein production and cellular protein expression in recombinant CHO cells subjected to osmotic and low culture temperature stress = 삼투압 및 저온 배양 stress에 의한 재조합 CHO 세포의 외래단백질 생산 및 세포 내 단백질 발현 변화 / Moon-Sue Lee.
저자명 Lee, Moon-Sue ; 이문수
발행사항 [대전 : 한국과학기술원, 2004].
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8015430

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DBS 04010

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초록정보

Hyperosmotic pressure has been suggested as an economical solution to increase the specific protein productivity (q). When we investigated the response of antibody-expressing rCHO cells ($CS13^*$ -1.00) subjected to hyperosmotic pressure, the positive relationship between immunoglobulin (Ig) mRNA level per cell and antibody productivity ($q_{Ab}$). It represented that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. While much work has been performed on the response of rCHO cells to hyperosmotic pressure, there are few reported studies on the response to hypoosmotic pressure. Considering the importance of rCHO cells as a mammalian host, we also investigated the effect of hypoosmotic pressure on rCHO cells ($CS13^*$ -1.00). At hypoosmotic pressure, $CS13^*$ -1.00 cells displayed decreased specific growth rate (μ) and $q_{Ab}$. When the medium osmolality was decreased from 300 mOsm/kg to 150 mOsm/kg, μ was decreased by 68% and qAb was increased by 128%. To understand the mechanism of enhanced $q_{Ab}$ resulting from hypoosmotic pressure, cellular responses of cells in the exponential phase of growth were observed at the transcription level. Total cytoplasmic RNA content per cell at 150 mOsm/kg was increased by 140%, compared with that at 300 mOsm/kg. On a per μ g RNA basis, Ig mRNA levels at 150 mOsm/kg were comparable to those at 300 mOsm/kg, indicating that hypoosmotic pressure did not lead to the preferential transcription of Ig mRNAs. Taken together, the data obtained here suggest that the increase in total RNA pool is primarily responsible for the enhanced $q_{Ab}$ of $CS13^*$-1.00 cells subjected to hypoosmotic pressure. To better understand intracellular responses to osmotic pressure of rCHO cells expressing an antibody, we have taken a proteomics approach. Using two-dimensional electrophoresis and mass spectrometry, a proteome profile of $CS13^*$-1.00 cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found 3 proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, were found to be up-regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up-regulation of these glycolytic enzymes. On the other hand, tubulin expression was down-regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Thus, the activation of these two glycolytic enzymes results in an increased conversion of glucose into ATP and pyruvate and thereby generates more metabolic energy. The increased metabolic energy may lead to enhanced $q_{Ab}$ under hyperosmotic pressure. Taken together, it shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells. Moreover, the effect of another environmental parameter, low culture temperature, was investigated using genomic and proteomic analysis. Although lowering culture temperature below 37℃ decreases μ , a number of studies demonstrate beneficial effects. In the previous study, low culture temperature enhanced q of EPO-expressing rCHO cells (LGE10-9-27). To better understand intracellular responses of low culture temperature (33℃), we have taken genomic and proteomic approaches. For transcriptome analysis, low temperature response in gene expression profile was investigated using commercially available rat and mouse cDNA microarrays. When we analyzed regulations of all known gene expressions by rat cDNA chip experiment, 299 and 2 genes were up- and down-regulated over 2-fold at 33oC. In the result of mouse cDNA chip experiment, 44 and 118 genes were up- and down-regulated over 2-fold at 33℃. Genomic analysis showed that low culture temperature could lead to change gene expression in the various cellular processes, such as metabolism, transport, signaling pathway. Simultaneously, the regulations of 8 genes were overlapped in two kinds of cDNA chips. Although the two microarray experiments were performed successfully, the correspondence between two microarray results was not observed. Restricted interpretation was achieved from the rat and mouse microarray results. Because limited gene expression, which has sequence homology with CHO transcriptome, was detectable using two commercial chips. Moreover, proteome analysis was carried out using 2-D PAGE in conjunction with MALDI-TOF technique. Out of over 800 spot, 19 protein spots were regulated. Among these spots, 11 spots were selected as up-regulated candidates, some of which function in the metabolism (aldehyde dehydrogenase, isovaleryl coenzyme A dehydrogenase, H(+)-transporting ATP synthase, and glyceraldehyde 3-phosphate dehydrogenase), translation (similar to elongation factor Tu), and chaperone activity (PDI A6 precursor and ERp29 precursor). The rest 8 spots were selected as down-regulated candidates. Down-regulated 3 spots were identified as proteins which function in cell growth and maintenance (cytoplasmic actin, tubulin β 5, and γ -actin). This study, for the first time, explores the feasibility of commercially available cDNA chip for CHO transcriptome analysis. Moreover, the comparison of mRNA transcriptome and proteome expression was achieved for a relatively large number of gene expressions. This combined approach can be helpful for the powerful understanding of cellular mechanism at once. Furthermore, it showed the preliminary possibility for application of high-throughput genomic and proteomic analysis on CHO cell culture engineering.

서지기타정보

서지기타정보
청구기호 {DBS 04010
형태사항 viii, 158 p. : 삽도 ; 26 cm
언어 영어
일반주기 Appendix : The genes showing more than 2-fold up and down regulation in (A)rat cDNA chip and (B) mouse cDNA chip
저자명의 한글표기 : 이문수
지도교수의 영문표기 : Gyun-Min Lee
지도교수의 한글표기 : 이균민
수록잡지명 : "Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure ". Biotechnology progress, volume 19 issue 6 , pp 1734 - 1741(2003)
학과명칭변경 : 생물과학과가 생명과학과로 변경됨
학위논문 학위논문(박사) - 한국과학기술원 : 생명과학과,
서지주기 Reference : p. 113-122
주제 PROTEOMICS
ANIMAL CELL
CHO CELL
OSMOLARITY
FOREIGN PROTEIN PRODUCTION
프로테오믹스
동물세포
CHO세포
삼투압
외래단백질 생산
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