The pcDNA™6 BioEase™ Gateway® Biotinylation System provides an easy, efficient method for expressing, purifying, and detecting biotinylated recombinant proteins. The pcDNA™6/BioEase™-DEST vector (Figure 1) incorporates a 72 amino acid sequence from K. pneumoniae that directs in vivo biotinylation of a specific lysine residue. This K. pneumoniae tag is recognized and efficiently biotinylated in vivo by a native mammalian system similar to the enzyme Biotin Protein Ligase (BPL) in E. coli (1). Proteins produced from the pcDNA™6/BioEase™-DEST vector are expressed as fusion proteins to this sequence. Biotinylated fusion proteins can be purified by streptavidin agarose, detected on western blots using streptavidin-HRP or streptavidin-AP conjugates, or visualized in mammalian cells by immunofluorescence with a streptavidin-FITC conjugate (Figure 2). In addition to the 72 amino acid K. pneumoniae sequence, the pcDNA™6/BioEase™-DEST vector includes the following features:

• attR sites for fast and efficient recombination with any attL-flanked Gateway® entry vector

• The CMV promoter for high-level expression of the recombinant fusion protein

• An enterokinase cleavage site for efficient removal of the N-terminal BioEase™ fusion tag