Location of Repository

A model system to study the effects of methylglyoxal on the yield and quality of tissue plasminogen activator produced by CHO cells

By 

Abstract

Graduation date: 2000In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced

Year: 1999
OAI identifier: oai:ir.library.oregonstate.edu:1957/33264
Provided by: ScholarsArchive@OSU

Suggested articles

Preview

Citations

  1. (1996). Methylglyoxal induces DNA modification: A model system of DNA mutagenesis.
  2. Short Protocols in Molecular Biology. New York: Greene Publishing Associates and doi
  3. (1983). Two forms of tissue-type plasminogen activator (tPA) differ at a single specific glycosylation site.
  4. (1985). Methylglyoxal induced DNA-protein crosslinks and cytoxicity in Chinese hamster ovary cells. doi
  5. (1995). In-situ removal of ammonium and lactate through electrical means for hybridoma cultures. doi
  6. (1996). Detection of Methylglyoxal as a Degradation Product of DNA and Deoxyribonucleotides Treated with Strong Acid. doi
  7. (1996). Method for determination of free extracellular and intracellular methylglyoxal in animal cells grown in culture. doi
  8. (1998). Evidence of high levels of methylglyoxal in Chinese hamster ovary cells. doi
  9. (1998). Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A Review.
  10. (1984). Effects of exogenous amines on mammalian cells, with particular reference to membrane flow.
  11. (1979). Rregulation of plasminogen activator secretion in mouse peritoneal macrophages. doi
  12. (1985). Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. doi
  13. (1994). Cultural and physiological factors affecting expression of recombinant proteins. doi
  14. (1994). Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture. doi
  15. (1985). Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.
  16. (1993). Human glyoxalase I CDN A cloning, expression and sequence similarity to glyoxalase I from Pseudomonai putida.
  17. (1995). cDNA cloning and characterization of human glyoxalase I isoforms from HT-1080 cells. doi
  18. (1997). Glycosylation of CHO-derived recombinant tPA produced under elevated pCO2. doi
  19. (1973). Reaction of methylglyoxal with nucleic acids. doi
  20. (1993). Production of tPA in recombinant CHO cells under oxygen limited conditiond. doi
  21. (1994). Binding and modification of proteins by methylglyoxal under physiological conditions. doi
  22. (1989). Molecular Cloning, A Laboratory Manual, Second edition. doi
  23. (1996). Ecdysone-Inducible Gene Expression in Mammailian Cells and Transgenic Mice. doi
  24. (1995). Identificaiton of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end-product. doi
  25. (1989). N-Glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. doi
  26. (1986). Purification and characterization of glyoxalase I from Pseudomonas putida. doi
  27. (1984). Acid-base catalysis of the elimination and isomerization reactions of triose phosphates. doi
  28. (1996). Optimized heterologous expression of the human zinc enzyme glyoxalase I.
  29. (1981). Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture.
  30. (1991). Growht limitation in hybridoma cell cultures" The role of inhibitory or toxic metabolites. doi
  31. (1994). Inhibitory substances secreted in cell culture media of a recombinant CHO and a hybridoma cell line. In: doi
  32. (1996). Pharmacology of methylglyoxal: Formation, modification of proteins and nucleic acids, and enzymatic detoxification A role in pathogenesis and antiproliferative chemotherapy. doi
  33. (1993). The glyoxalase system in health and disease. doi
  34. (1990). The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life.
  35. (1998). Modification of the glyoxalase system in human red blood cells by glucose in vitro.
  36. (1989). Cytogenic response of 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes. doi
  37. (1986). Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology. In: doi
  38. (1984). Characterization studies on human melanoma cell tissue plasminogen activator. BioITechnology doi
  39. (1988). Amplified expression oconstructs for human tissue-type plasminogen activator in Chinese hamster ovary cells:instability in the absence of selective pressure. doi
  40. (1995). Molecular characteristics of methylglyoxalmodifies bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins. doi

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.