Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.
The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.
Daniels-McQueen
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Department of Biology, Washington University, St. Louis, MO 63130.
Goessling
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Thach
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D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics*; D002460:Cell Line; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000235:genetics; D005293:Ferritins / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D051379:Mice; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004247:DNA; D005293:Ferritins