Production of the chimerical plasminogen activator K2tu-PA in CHO cells
| Autor: | Jessica Hamerman, Roland Widmer, Fred A.M. Asselbergs |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
DNA
Complementary Recombinant Fusion Proteins Molecular Sequence Data Cytomegalovirus Bioengineering CHO Cells Biology Transfection Applied Microbiology and Biotechnology Mice Cricetinae Gene expression Animals Promoter Regions Genetic Enhancer Genes Immediate-Early Gene Expression vector Base Sequence T-plasminogen activator Chinese hamster ovary cell Gene Amplification Promoter General Medicine Urokinase-Type Plasminogen Activator Molecular biology Tetrahydrofolate Dehydrogenase Enhancer Elements Genetic Tissue Plasminogen Activator Rabbits Plasminogen activator Biotechnology |
| Zdroj: | Journal of Biotechnology. 42:221-233 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/0168-1656(95)00082-2 |
| Popis: | Development of a CHO cell-based production system for the hybrid plasminogen activator K2tu-PA is described. Using the major immediate-early promoter of mouse cytomegalovirus (MCMV) transient and stable expression levels were 3-10-fold higher than those obtained with several other strong promoters. Splicing and polyadenylation signals from the rabbit beta-globin gene were used downstream of the DNA segment coding for K2tu-PA. The strong enhancer moiety of the MCMV promoter also stimulated strongly the promoter of the dihydrofolate reductase (DHFR) gene, placed adjacently for selection/gene amplification purposes. One construct with opposing K2tu-PA and DHFR RNA transcripts yielded the highest expression level with a single copy of the plasmid, but K2tu-PA expression was consistently lost after amplification of such genes, possibly as a result of the formation of antisense RNA. With other constructs, K2tu-PA production leveled off at 6.5 micrograms per million cells per day despite a high gene copy number. This was due to a combination of inefficient mRNA translation and mRNA instability, caused by elements from the untranslated portions of tissue-type and urokinase-type plasminogen activator cDNA which were included in the expression vector. After elimination of these inhibitory DNA segments, 4-5-times higher expression levels were reached. |
| Databáze: | OpenAIRE |
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