2024-03-29T09:23:28Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/813102021-12-27T16:05:46Zcom_10261_105com_10261_1col_10261_358
DIGITAL.CSIC
author
Buj, Raquel
author
Iglesias, Noa
author
Planas, Anna M.
author
Santalucía, Tomàs
funder
CSIC - Unidad de Recursos de Información Científica para la Investigación (URICI)
2013-09-03T09:28:35Z
2013-09-03T09:28:35Z
2013-08-20
BMC Molecular Biology 14(1): 18 (2013)
1471-2199
http://hdl.handle.net/10261/81310
10.1186/1471-2199-14-18
23957834
[Background]: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading
frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted
to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.
[Results]: We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to
the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional
modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s
component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.
[Conclusions]: We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.
eng
openAccess
Gateway cloning
Fusion protein
Combinatorial
Fluorescent protein
Epitope tag
A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector
artículo
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URL
https://digital.csic.es/bitstream/10261/81310/1/A%20plasmid%20toolkit.pdf
File
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A plasmid toolkit.pdf
URL
https://digital.csic.es/bitstream/10261/81310/4/A%20plasmid%20toolkit.pdf.txt
File
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A plasmid toolkit.pdf.txt