原创力文档an intein with genetically selectable markers provides a new approach to internally label proteins with gfpintein与选择标记基因提供了一种新的方法在内部标签蛋白gfp.pdfVIP
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an intein with genetically selectable markers provides a new approach to internally label proteins with gfpintein与选择标记基因提供了一种新的方法在内部标签蛋白gfp
Ramsden et al. BMC Biotechnology 2011, 11:71
/1472-6750/11/71
RESEARCH ARTICLE Open Access
An intein with genetically selectable markers
provides a new approach to internally label
proteins with GFP
*
Richard Ramsden, Luther Arms, Trisha N Davis and Eric GD Muller
Abstract
Background: Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the
process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently
have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity
are independent and distinct domains in the folded structure. We show here that other biochemical activities can
be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can
provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-
terminal extein to create a cassette to introduce GFP within the interior of a targeted protein.
Results: The Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1) aminoglycoside
phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3) hygromycin B
phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost
endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency
was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either
G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast
genetic background. DNA encoding the marked
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