AviTag™ ORF cDNA clones, Protein Biotinylation

生物素连接酶 (Biotin Ligase)由birA基因编码所得，能活化生物素形成生物素酰 -5'-腺苷酸，将生物素特异地转移到生物素受体标签蛋白（如AviTag 融合蛋白）上，使标签蛋白生物素化，也可用作生物素操纵子阻碍物。又名为：生物素操纵子阻碍蛋白(biotin operon repressor protein)、birA、生物素酶合成酶(biotin holoenzyme synthetase)、生物素-乙酰辅酶a羧化酶合成酶(biotin-[acetyl-CoA carboxylase] synthetase)。

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生物素连接酶的相关菌株

AVB 100菌株

AVB100是一种大肠杆菌E. coli K12菌株 [MC1061 araD139 delta(ara-leu)7696 delta(lac)l74 galU galK hsdR2(rK-mK+) mcrB1 rpsL(Strr)]。该菌株的染色体带有稳定转入的birA基因。

在该菌株中，BirA蛋白的过表达可由阿拉伯糖诱导所得。稳定转入的birA基因不需使用抗生素维持。在AVB100使用IPTG诱导的载体如Avidity's pAC、pAN、pATN 和pATC AviTag载体，可独立调控目的基因及BirA的表达水平。该菌株仅限个人研究使用，不可分销，使用前请阅读产品的相关许可证明。

AVB99菌株

AVB99是一种带有pACYC184质粒的大肠杆菌E.coli (XL1-Blue)，该质粒包含IPTG诱导的birA基因，可过表达生物素连接酶(pBirAcm)，是一个低拷贝数的质粒。培养含该质粒的菌株可使用10 µg/mL浓度的氯霉素。

AVB101菌株

AVB101是大肠杆菌E. coli 的B菌株(hsdR, lon11, su1A1)，内含pACYC184质粒，该质粒含有IPTG诱导的birA基因，可过表达生物素连接酶(pBirAcm)。该菌株生长快速且健康，不含添加OmpT或Lon蛋白酶，是理想的蛋白表达工具菌株。

生物素连接酶及AviTag蛋白的表达均由IPTG (1 mM)诱导。诱导阶段必须添加生物素，添加至其终浓度为50 µM。pAN及pAC载体需添加氨苄青霉素维持，抗生素浓度建议为100 µg/mL。

Avidity's AVB99及AVB101的质粒特性：

质粒骨架为pACYC184；
其中1.3 Kb片段含有插入了EcoRV位点的lacIq(因此可干扰tet^R基因)；
BirA由乳糖启动子操纵子调控表达；
rrnB终止子结构位于laqIq区域，可破坏pACYC184的XbaI位点。

Example of in vitro biotinylation of AviTag recombinant proteins

Figure1. Extracts from 293T cells transfected with the AviTag eGFP or AviTag-IkBαexpression plasmid and untransfected controls were mixed with purified biotin ligase, biotin (50M) and ATP (10mM) as indicated in the figure. After incubation at 30^.C (lanes 1-7, 9-13) or 4^.C (lanes 8, 14) for 30 minutes, the mixtures were resolved by SDS-PAGE. Biotinylated proteins were visualized on a western blot with streptavidin-HRP conjugate and chemiluminescent substrate (Pierce).

Example of in vivo biotinylation of AviTag recombinant proteins

Figure2. AviTag-eGFP or AviTag- IkBα expression plasmids were transfected into 293T cells alone or with co-transfected biotin ligase. Lysates were prepared 13 hours. Extracts from after transfection and resolved on a 12% SDS-PAGE gel. Biotinylated proteins were visualized on a western blot with streptavidin-HRP conjugate and chemiluminescent substrate (Pierce).

Example of precipitation of biotinylated AviTag recombinant proteins with streptavidin beads

Figure3. 293T cells were transfected with different combinations of AviTag expression plasmids and biotin ligase, as indicated in the figure. Lysates were prepared 24 hours after transfection and incubated with streptavidin beads for 8 hours. Suspensions were centrifuged and pelleted beads were washed 3 times. Cell lysates (L), supernatants (S) and pelleted biotinylated protein bound to streptavidin beads (B) were resolved by SDS-PAGE. Biotinylated proteins were visualized on a western blot with streptavidin-HRP conjugate and chemiluminescent substrate (Pierce).

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购买	货号	描述	规格	价格
Biotinylation Strains
	AVB101	Efficient Biotinylation E. coli B Strain AVB101	1 vial, containing 0.5 mL of glycerol stock	￥2400
	AVB100	Efficient Biotinylation E. coli K12 Strain AVB100	1 vial, containing 0.5 mL of glycerol stock	￥2400
Avidity Vectors
	PAN4	Biotin AviTag™ Vectors (N-terminal)	10 μg	￥1584 each
	PAN5	Biotin AviTag™ Vectors (N-terminal)	10 μg	￥1584 each
	PAN6	Biotin AviTag™ Vectors (N-terminal)	10 μg	￥1584 each
	PAC4	Biotin AviTag™ Vectors (C-terminal)	10 μg	￥1586 each
	PAC5	Biotin AviTag™ Vectors (C-terminal)	10 μg	￥1586 each
	PAC6	Biotin AviTag™ Vectors (C-terminal)	10 μg	￥1586 each

参考文献

2017

Daiss, J.L., et al. (2017). DIAGNOSTIC DEVICE AND METHOD FOR DETECTION OF STAPHYLOCOCCUS INFECTION. United States Patent Application 20170023569 [AviTag™ sequence]

AviTag™ 生物素相关试剂