title: A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.
abstract: 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.
authors: Department of Brain Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona (IIBB)-Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.
authors: Raquel
authors: R
authors: Buj
authors: Noa
authors: N
authors: Iglesias
authors: Anna M
authors: AM
authors: Planas
authors: Tomàs
authors: T
authors: Santalucía
mesh_terms: D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase
references: Trends Biotechnol. 2012 Jan;30(1):8-16
references: 21924508
references: PLoS One. 2012;7(10):e47656
references: 23082188
references: Dev Dyn. 2007 Nov;236(11):3088-99
references: 17937395
references: Protein Eng. 1998 Dec;11(12):1211-7
references: 9930670
references: BMC Mol Biol. 2012 Sep 20;13:30
references: 22994806
references: J Mol Biol. 1990 May 5;213(1):167-86
references: 2187098
references: Genome Res. 2004 Oct;14(10B):2001-9
references: 15489318
references: Yeast. 2007 Oct;24(10):913-9
references: 17583893
references: Nat Biotechnol. 2007 Jun;25(6):663-8
references: 17486083
references: Curr Opin Struct Biol. 2011 Jun;21(3):398-403
references: 21497084
references: Nucleic Acids Res. 2006;34(19):5528-40
references: 17023487
references: Protein Eng Des Sel. 2007 Jul;20(7):315-8
references: 17575283
references: J Biol Chem. 2007 Mar 2;282(9):6823-32
references: 17197703
references: Genomics. 2007 Mar;89(3):307-15
references: 17207965
references: Eur J Nucl Med Mol Imaging. 2013 Feb;40(3):426-38
references: 23135322
references: J Neurosci. 2008 Aug 13;28(33):8246-56
references: 18701687
references: Nat Methods. 2005 Dec;2(12):905-9
references: 16299475
references: PLoS One. 2011 Feb 18;6(2):e16765
references: 21364738
references: Biochemistry. 2011 Feb 1;50(4):437-9
references: 21175224
references: Trends Biotechnol. 2005 Apr;23(4):199-207
references: 15780712
references: EMBO Rep. 2000 Sep;1(3):287-92
references: 11256614
references: BMC Mol Biol. 2006 Dec 06;7:46
references: 17150110
references: J Biol Chem. 1999 Jun 25;274(26):18524-35
references: 10373461
references: Science. 2005 Sep 2;309(5740):1559-63
references: 16141072
references: Curr Opin Biotechnol. 2009 Dec;20(6):616-22
references: 19850467
references: Curr Opin Chem Biol. 2004 Feb;8(1):20-5
references: 15036152
references: PLoS One. 2011;6(9):e24531
references: 21931740
references: PLoS One. 2008 Jan 30;3(1):e1528
references: 18231609
references: J Biol Chem. 1999 Dec 31;274(53):37941-9
references: 10608861
references: PLoS One. 2008;3(11):e3647
references: 18985154
references: Expert Opin Drug Discov. 2012 May;7(5):371-4
references: 22468854
references: Genome Res. 2004 Oct;14(10B):2169-75
references: 15489340
references: Nature. 2010 Dec 16;468(7326):968-72
references: 21107320
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Aug 1;68(Pt 8):878-82
references: 22869113
references: Appl Environ Microbiol. 2010 Mar;76(5):1633-41
references: 20081001
references: Genome Res. 2004 Oct;14(10B):2111-20
references: 15489333
references: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16899-903
references: 12477932
references: Protein Eng Des Sel. 2007 Jul;20(7):309-13
references: 17332010
references: Genome Res. 2000 Nov;10(11):1788-95
references: 11076863
references: Biochem J. 2009 Mar 15;418(3):567-74
references: 19143658
references: Development. 2011 Dec;138(24):5451-8
references: 22110059
references: J Biotechnol. 2008 Sep 10;136(3-4):103-12
references: 18640160
references: PLoS One. 2007 Sep 19;2(9):e916
references: 17878951
references: BMC Biotechnol. 2007 Jul 30;7:45
references: 17663785
references: PLoS One. 2011;6(7):e21622
references: 21750718