title: Production of the chimerical plasminogen activator K2tu-PA in CHO cells.
abstract: 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.
authors.affiliation: CIBA-GEIGY Ltd., Pharma Research Department, Basle, Switzerland.
authors.forename: F A
authors.initials: FA
authors.lastname: Asselbergs
authors.forename: J
authors.initials: J
authors.lastname: Hamerman
authors.forename: R
authors.initials: R
authors.lastname: Widmer
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic; D005784:Gene Amplification; D017781:Genes, Immediate-Early; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator