A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome

Cited 4 time in webofscience Cited 0 time in scopus
  • Hit : 318
  • Download : 0
Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor. (C) 2012 Elsevier Inc. All rights reserved.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2012-07
Language
English
Article Type
Article
Keywords

ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; CHROMOSOMAL GENES; BIOSYNTHETIC GENE; EXPRESSION; LEUCINE; SEQUENCE; DELETION; PROTEIN; CLUSTER

Citation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, v.424, no.1, pp.22 - 27

ISSN
0006-291X
DOI
10.1016/j.bbrc.2012.06.022
URI
http://hdl.handle.net/10203/102775
Appears in Collection
BS-Journal Papers(저널논문)
Files in This Item
There are no files associated with this item.
This item is cited by other documents in WoS
⊙ Detail Information in WoSⓡ Click to see webofscience_button
⊙ Cited 4 items in WoS Click to see citing articles in records_button

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0