Invitrogen™ Champion™ pET104 BioEase™ Gateway™ Biotinylation System

Invitrogen™ Champion™ pET104 BioEase™ Gateway™ Biotinylation System

The Champion™ pET104-DEST BioEase™ Gateway® Expression System provides an easy, efficient method for expressing, purifying, and detecting biotinylated recombinant proteins. The Champion™ pET104-DEST vector (Figure 1) incorporates a 72 amino acid sequence from K. pneumoniae that directs in vivo biotinylation of a specific lysine residue. This tag is recognized and efficiently biotinylated by the enzyme Biotin Protein Ligase (BPL) encoded by the birA gene in E. coli(1). Proteins produced in the Champion™ pET104-DEST vector are expressed as fusion proteins to this sequence. Biotinylated fusion proteins can be purified on streptavidin agarose or detected on western blots using streptavidin-HRP (Figure 2) or streptavidin-AP conjugates.

Features

• attR sites for efficient recombination with any attL-flanked Gateway® entry vector
• The T7lac bacteriophage promoter for high-level expression of the recombinant fusion protein
• An enterokinase cleavage site for removal of the N-terminal BioEase™ fusion tag

Contents 

• 6 flg of the Champion™ pET104-DEST vector
• 10 µg of the Champion™ pET104/GW/lacZ control vector
• twenty-one 50-µl aliquots of BL21 Star™(DE3) One Shot¤ competent cells
• S.O.C. medium
• pUC19 control DNA.