生物素酰化重组蛋白表达系统 哺乳动物表达载体pcDNA6/BioEase表达生物素酰化的融合蛋白，提供了一种简便、高效的检测和纯化手段。BioEase标签是含72个氨基酸残基的肽段，体内的生物素连接酶系统（ Biotin Protein Ligase），使这段肽链上特定的赖氨酸残基生物素酰化。生物素酰化的融合蛋白可以用链霉亲和素琼脂糖珠进行纯化。
The pcDNA6-BioEase Gateway Biotinylation System provides an easy, efficient method for expressing, purifying, and detecting biotinylated recombinant proteins. The pcDNA6/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 pcDNA6/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 pcDNA6/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
Gateway哺乳动物表达载体选择指南：
pDEST12.2 不带标签
pDEST26 N-His标签 G418筛选
pDEST27 N-GST标签 G418筛选
pDEST40 C-His标签、C-V5标签 G418筛选
pcDNA-DEST40 C-His标签、C-V5标签 G418筛选
pcDNA-DEST47 C-绿色荧光蛋白 G418筛选
pcDNA-DEST53 N-绿色荧光蛋白 G418筛选
pcDNA3.2/V5-DEST C-V5标签 G418筛选
pcDNA6.2/V5-DEST C-V5标签 Blasticidin S筛选
pcDNA6.2/N-YFP-DEST N-黄色荧光蛋白 Blasticidin S筛选
pcDNA6.2/N-EmGFP-DEST N-绿色荧光蛋白 Blasticidin S筛选
pcDNA6.2/C-YFP-DEST C-黄色荧光蛋白 Blasticidin S筛选
pcDNA6.2/C-EmGFP-DEST C-绿色荧光蛋白 Blasticidin S筛选
pcDNA6.2/BioEase-DEST N-生物素 Blasticidin S筛选
