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Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.
- Source :
-
Journal of cell science [J Cell Sci] 2017 Mar 01; Vol. 130 (5), pp. 1003-1015. Date of Electronic Publication: 2017 Jan 12. - Publication Year :
- 2017
-
Abstract
- GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe , we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.<br /> (© 2017. Published by The Company of Biologists Ltd.)
- Subjects :
- Base Sequence
Genes, Reporter
Genetic Vectors metabolism
Luciferases metabolism
Promoter Regions, Genetic genetics
Protein Binding
Protein Transport
Recombinant Fusion Proteins metabolism
Schizosaccharomyces pombe Proteins genetics
Schizosaccharomyces pombe Proteins metabolism
Spindle Pole Bodies metabolism
Cytological Techniques methods
Green Fluorescent Proteins metabolism
Schizosaccharomyces metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1477-9137
- Volume :
- 130
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of cell science
- Publication Type :
- Academic Journal
- Accession number :
- 28082423
- Full Text :
- https://doi.org/10.1242/jcs.198457