New donor vector for generation of histidine-tagged fusion proteins using the Gateway Cloning System.
An optimized donor/shuttle vector, pENTR-His-ccdB, was generated that readily produces a histidine-tagged recombinant protein in multiple expression systems using Gateway Technology. In the current Gateway System, six histidines and the tobacco etch virus protease cleavage site are encoded upstream of the attR1 recombination site such that the histidine-tagged destination/expression vector adds 15 residues to the amino-terminus of recombinant proteins. Our new vector introduces the histidine tag at the donor level and places the multiple cloning sites within the attL recombination sites producing cleavable histidine-tagged proteins with a short, neutral linker of five residues. Two histidine-tagged clones were produced and fusion proteins expressed using the newly engineered vector.
Parr
Rebecca D
RD
Department of Pathobiology, Texas A&M University, MS 4467, VMS 119, College Station, TX 77843, USA.
Ball
Judith M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D051242:Caveolin 1; D022461:Caveolins; C068356:CcdB protein, Plasmid F; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D006639:Histidine; D012321:DNA-Directed RNA Polymerases