Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27KIP1, a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27KIP1 plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27KIP1 was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27KIP1 and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn2+, Cu2+, or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Jan 16 2001|
All Science Journal Classification (ASJC) codes
- Cell cycle
- Coupled transcription