Applied Microbiology and Biotechnology

, Volume 89, Issue 6, pp 1917–1928 | Cite as

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

Genomics, Transcriptomics, Proteomics

Abstract

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (qAb, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.

Keywords

CHO cells Proteomics Hydrolysates Cell growth Productivity 

References

  1. Baik JY, Lee MS, An SR, Yoon SK, Joo EJ, Kim YH, Park HW, Lee GM (2006) Initial transcriptome and proteome analyses of low culture temperature-induced expression in CHO cells producing erythropoietin. Biotechnol Bioeng 93:361–371CrossRefGoogle Scholar
  2. Baik JY, Joo EJ, Kim YH, Lee GM (2008) Limitations to the comparative proteomic analysis of thrombopoietin producing Chinese hamster ovary cells treated with sodium butyrate. J Biotechnol 133:461–468CrossRefGoogle Scholar
  3. Chun BH, Kim JH, Lee HJ, Chung N (2007) Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture. Bioresour Technol 98:1000–1005CrossRefGoogle Scholar
  4. Durocher Y, Butler M (2009) Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20:700–707CrossRefGoogle Scholar
  5. Filipenko NR, Waisman DM (2001) The C terminus of annexin II mediates binding to F-actin. J Biol Chem 276:5310–5315CrossRefGoogle Scholar
  6. Filipenko NR, MacLeod TJ, Yoon CS, Waisman DM (2004) Annexin A2 is a novel RNA-binding protein. J Biol Chem 279:8723–8731CrossRefGoogle Scholar
  7. Fouassier L, Rosenberg P, Mergey M, Saubaméa B, Clapéron A, Kinnman N, Chignard N, Jacobsson-Ekman G, Strandvik B, Rey C, Barbu V, Hultcrantz R, Housset C (2009) Ezrin-radixin-moesin-binding phosphoprotein (EBP50), an estrogen-inducible scaffold protein, contributes to biliary epithelial cell proliferation. Am J Pathol 174:869–880CrossRefGoogle Scholar
  8. Franek F, Hohenwarter O, Katinger H (2000) Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures. Biotechnol Prog 16:688–692CrossRefGoogle Scholar
  9. Gachet Y, Tournier S, Lee M, Lazaris-Karatzas A, Poulton T, Bommer UA (1999) The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle. J Cell Sci 112:1257–1271Google Scholar
  10. Goode BL, Drubin DG, Barnes G (2000) Functional cooperation between the microtubule and actin cytoskeletons. Curr Opin Cell Biol 12:63–71CrossRefGoogle Scholar
  11. Grillberger L, Kreil TR, Nasr S, Reiter M (2009) Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells. Biotechnol J 4:186–201CrossRefGoogle Scholar
  12. Guo W, Xu H, Chen J, Yang Y, Jin JW, Fu R, Liu HM, Zha XL, Zhang ZG, Huang WY (2007) Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1. Mol Cell Biochem 295:167–177CrossRefGoogle Scholar
  13. Hakimelahi S, Parker HR, Gilchrist AJ, Barry M, Li Z, Bleackley RC, Pasdar M (2000) Plakoglobin regulates the expression of the anti-apoptotic protein BCL-2. J Biol Chem 275:10905–10911CrossRefGoogle Scholar
  14. Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Park S, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D (2000) The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology 32:157–167CrossRefGoogle Scholar
  15. Kim SH, Lee GM (2009) Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Appl Microbiol Biotechnol 83:639–648CrossRefGoogle Scholar
  16. Kim JY, Sing HJ, Lim HJ, Shin MG, Kim JS, Kim HJ, Kim BY, Lee SW (2008) Platelet factor-4 is an indicator of blood count recovery in acute myeloid leukemia patients in complete remission. Mol Cell Proteomics 7:431–441Google Scholar
  17. Kim JY, Kim YG, Baik JY, Joo EJ, Kim YH, Lee GM (2010) A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells. Biotechnol Prog 26:246–251Google Scholar
  18. Koo BS, Lee DY, Ha HS, Kim JC, Kim CW (2005) Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis. J Proteome Res 4:719–724CrossRefGoogle Scholar
  19. Kumble KD, Iversen PL, Vishwanatha JK (1992) The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides. J Cell Sci 101:35–41Google Scholar
  20. Lee MS, Kim KW, Kim YH, Lee GM (2003) Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure. Biotechnol Prog 19:1734–1741CrossRefGoogle Scholar
  21. Li J, Huang Z, Sun X, Yang P, Zhang Y (2006) 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. Enzyme Microb Technol 38:372–380CrossRefGoogle Scholar
  22. Liu Z, Ahn JY, Liu X, Ye K (2006) Ebp1 isoforms distinctively regulate cell survival and differentiation. Proc Natl Acad Sci USA 103:10917–10922CrossRefGoogle Scholar
  23. Moiseeva EP, Javed Q, Spring EL, de Bono DP (2000) Galectin 1 is involved in vascular smooth muscle cell proliferation. Cardiovasc Res 45:493–502CrossRefGoogle Scholar
  24. Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T (1997) Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultures. Cytotechnology 23:47–54CrossRefGoogle Scholar
  25. O'Callaghan PM, James DC (2008) Systems biotechnology of mammalian cell factories. Brief Funct Genomic Proteomic 7:95–110CrossRefGoogle Scholar
  26. Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, Durocher Y (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84:332–342CrossRefGoogle Scholar
  27. Powis G, Mustacich D, Coon A (2000) The role of the redox protein thioredoxin in cell growth and cancer. Free Radic Biol Med 29:312–322CrossRefGoogle Scholar
  28. Renard JM, Spagnoli R, Mazier C, Salles MF, Mandine E (1988) Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotechnol Lett 10:91–96CrossRefGoogle Scholar
  29. Schlaeger EJ (1996) The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J Immunol Methods 194:191–199CrossRefGoogle Scholar
  30. Shen CF, Kiyota T, Jardin B, Konishi Y, Kamen A (2007) Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production. Cytotechnology 54:25–34CrossRefGoogle Scholar
  31. Shimizu K, Chen W, Ashique AM, Moroi R, Li YP (2003) Molecular cloning, developmental expression, promoter analysis and functional characterization of the mouse CNBP gene. Gene 307:51–62CrossRefGoogle Scholar
  32. Sung YH, Lim SW, Chung JY, Lee GM (2004) Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 63:527–536CrossRefGoogle Scholar
  33. Van Dyk DD, Misztal DR, Wilkins MR, Mackintosh JA, Poljak A, Varnai JC, Teber E, Walsh BJ, Gray PP (2003) Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line. Proteomics 3:147–156CrossRefGoogle Scholar
  34. Vié N, Copois V, Bascoul-Mollevi C, Denis V, Bec N, Robert B, Fraslon C, Conseiller E, Molina F, Larroque C, Martineau P, Del Rio M, Gongora C (2008) Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells. Mol Cancer 7:e14CrossRefGoogle Scholar
  35. Voglauer R, Chang MW, Dampier B, Wieser M, Baumann K, Sterovsky T, Schreiber M, Katinger H, Grillari J (2006) SNEV overexpression extends the life span of human endothelial cells. Exp Cell Res 312:746–759CrossRefGoogle Scholar
  36. Yang JP, Hori M, Takahashi N, Kawabe T, Kato H, Okamoto T (1999) NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2. Oncogene 18:5177–5186CrossRefGoogle Scholar
  37. Yee JC, de Leon GM, Philp RJ, Yap M, Hu WS (2008) Genomic and proteomic exploration of CHO and hybridoma cells under sodium butyrate treatment. Biotechnol Bioeng 99:1186–1204CrossRefGoogle Scholar
  38. Zhang AH, Rao JN, Zou T, Liu L, Marasa BS, Xiao L, Chen J, Turner DJ, Wang JY (2007) p53-dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion. Am J Physiol Cell Physiol 293:379–389CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Biological Sciences and Graduate School of Nanoscience & Technology (WCU)DaejonSouth Korea
  2. 2.Division of Mass Spectrometry Research, Korea Basic Science InstituteCheongwon-GunSouth Korea
  3. 3.Graduate School of Analytical Science and TechnologyChungnam National UniversityYuseong-GuSouth Korea

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