常见蛋白质标签总结.doc

/bbs/home.php?mod=space&uid=34800&do=blog&id=38530常见蛋白质标签总结（Flag、HA、cMyc、CBP等）Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.一、氨基酸标签（含小肽标签）A stretch of amino acids is added to the protein and enables the recovery of the labelled protein by its unique affinity. Usually its easiest to add the tag to either end of the protein to ensure its accessibility and not to disturb the protein folding.1. 组氨酸标签（His tag）一般为6个组氨酸，用Ni2+ （Cu2+）亲和层析纯化2. FLAG tag ：N-DYKDDDDK-C ，recovered with specific antibody3. HA tag： an epitope derived from the Influenza protein haemagglutinin (HA，禽流感病毒血凝素)，e.g. N-YPYDVPDYA-C，recovery with an HA antibody4. MYC tag： an epitope derived from the human proto-oncoprotein MYC，e.g. N-ILKKATAYIL-C, N-EQKLISEEDL-C，recovery with an MYC antibody5. SBP tag：Streptavidin Binding Peptide，链霉亲合素结合肽，38 amino acid tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)，更多参考在Sigma6. CBP tag：钙调蛋白结合肽（CBP; 26aa）钙调蛋白结合肽与钙调素结合是Ca2+依赖的，这种结合不受标签所处的位置影响（N端和C端均可），在中性pH条件下使用2mM EGTA可以很方便的将目标蛋白洗脱下来。这一系统有如下优点：1 特异性很高，因为大肠杆菌没有可以和钙调素结合的蛋白；2 与His标签相比可以在强还原性条件下纯化。7. 纤维素结合肽（CBD）： 能与纤维素介质特异性的结合，可以在温和的条件下洗脱（乙二醇或低盐条件），pET CBD 载体含有纤维素结合肽（CBD）的序列，可方便构建。二、蛋白质标签Rather than adding only a few amino acids a whole protein is fused to the protein to be purified or detected. The affinity of the attached protein enables the recovery of the artificial fusion protein. As for the peptides, the protein tag is added to either end of the target protein.1. GST tag： the small glutathione-S-transferase (GST; 26 kDa)，recovery by affinity to substrate glutathione bound to a column, e.g. glutathione sepharose2. MBP tag：麦芽糖结合蛋白（MBP; 40kDa）载体：pMAL3. IMPACT：Intein-Mediated Purification with an affinity Chitin-binding Tag（几丁质结合肽）4. 硫氧还蛋白：Thioredoxins are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange.5. Protein A： a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has found use in biochemical research because of its ability to bind immunoglobulins. It binds proteins from many of mammalian species, most notably IgGs. It binds with the Fc region of immunoglobulins through interaction with the heavy chain. 6. Protein A/G：a recombinant fusion protein that combines IgG binding domains of both Protein A and Protein G. Protein A/G contains four Fc binding domains from Protein A and two from Protein G, yielding a final mass of 50,460 daltons. The binding of Protein A/G is less dependent upon pH than Protein A, but otherwise has the additive properties of Protein A and G. Protein A/G binds to all subclasses of human IgG, making it useful for purifying polyclonal or monoclonal IgG antibodies whose subclasses have not been determined. In addition, it binds to IgA, IgE, IgM and (to a lesser extent) IgD. Protein A/G also binds to all subclasses of mouse IgG but does not bind mouse IgA, IgM or serum albumin.7. GFP：绿色荧光蛋白（GFP; 26.9kDa）GFP and its variants are the most commonly used fluorescence tags.三、化学标签biotin - streptavidin （生物素 - 链霉亲合素）linking amino acids, often lysines, to cofactor and vitamin biotin purification by tight binding to streptavidin-agarose or -beads.Streptavidin is a 53,000 dalton tetrameric protein purified from the bacterium Streptomyces avidinii. It finds wide use in molecular biology through its extraordinarily strong affinity for the vitamin biotin; the dissociation constant (Kd) of the biotin-streptavidin complex is on the order of 10-15 mol/L, ranking among one of the strongest