Come in from the Cold

For high-throughput screening and crystallography studies, it is often necessary to express large quantities of protein--a process that is complicated if the protein is toxic to the cells in which it is expressed. To combat this difficulty, transcription of the gene of interest is repressed while the cells are grown to a high density and then induced by the addition of reagents such as IPTG or tetracycline. Although chemical induction has been successfully used to regulate protein expression for

Written byAileen Constans

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Addressing this problem, Cytos Biotechnology AG of Zürich-Schlieren, Switzerland, has designed an inducible expression vector that uncouples transcription from induction. The system uses the vector pCytTS, which is derived from the alphavirus DNA vector p987SinRep96. The vector contains the gene for a temperature-sensitive RNA-dependent RNA replicase upstream of a "subgenomic promoter" and a multiple cloning region into which researchers clone their gene of interest. The replicase gene includes two point mutations, which render it inactive at 37ºC owing to improper folding. When the temperature is reduced to less than 35ºC, the replicase becomes activated. This active replicase replicates the full-length transcript containing both the replicase and cloned genes in the cytoplasm, but it also begins to produce excess mRNA encoding only the gene of interest, initiating transcription at the subgenomic promoter. According to business development director Claudine Blaser, maximum expression occurs at 30ºC, and induction is more than 10,000-fold--a level ...

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