Expression of Recombinant Human Insulin in Chinese Hamster Ovary Cells is Complicated by Intracellular Insulin-Degrading Enzymes

Autor: M.J. Sleigh, Peter P. Gray, Stephen C. Pak, Sybille Marie Hunt
Rok vydání: 2006
Předmět:
Zdroj: New Developments and New Applications in Animal Cell Technology ISBN: 0792350162
DOI: 10.1007/0-306-46860-3_10
Popis: To investigate the role of insulin degrading enzymes in heterologous insulin gene expression, Chinese hamster ovary (CHO) cells were transfected with a mammalian expression vector carrying the cDNA for the human pre-proinsulin under the control of the RSV promoter. Stable transfectants were isolated and characterised in regard to insulin transcription, translation, processing and secretion. Levels of insulin expressed by these cells were extremely low, peaking at around 5–10 ng/106 cells/24 hours. Analysis of the secreted product indicated inefficient processing with less than 10% of the total product being fully processed. [125I]-Insulin degradation assays revealed insulin degrading activity in the cytosol and in the conditioned medium of CHO cells. In both cases the insulin degrading activity was significantly inhibited by the addition of bacitracin, a peptide antibiotic and an inhibitor of insulin degrading enzyme (IDE). No noticeable effects were seen with the addition of a general protease inhibitor, PMSF. When transfected CHO cultures were supplemented with small quantities of bacitracin insulin expressionimproved considerably. These results combined support the hypothesis that IDEs are directly involved in inhibiting overexpression of recombinant human insulin in transfected CHO cells.
Databáze: OpenAIRE
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