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1、常见蛋白质标签总结 2008-12-08 22:06Protein 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
2、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.组氨酸标签（His tag）一般为 6 个组氨酸，用 Ni2+ （Cu2+）亲和层析纯化 FLAG tag ：N-DYK
3、DDDDK-C ，recovered with specific antibody HA tag： an epitope derived from the Influenza protein haemagglutinin (HA，禽流感病毒血凝素)，e.g. N-YPYDVP-C，recovery with an HA antibody MYC tag： an epitope derived from the human proto-oncoprotein MYC，e.g. N-ILKKATAYIL-C, N-EQKLISEEDL-C，recovery with an MYC antibody
4、 SBP tag：Streptavidin Binding Peptide，链霉亲合素结合肽，38 amino acid tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)，更多参考在 Sigma 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。 纤维素
5、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 p
6、eptides, the protein tag is added to either end of the target protein.GST tag： the small glutathione-S-transferase (GST; 26 kDa)，recovery by affinity to substrate glutathione bound to a column, e.g. glutathione sepharose MBP tag：麦芽糖结合蛋白（MBP; 40kDa）载体：pMAL IMPACT：Intein-Mediated Purification with an
7、affinity Chitin-binding Tag（几丁质结合肽） 硫氧还蛋白：Thioredoxins are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Protein A： a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. I
8、t 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. Protein A/G：a recombinant fusion protein that combines
9、 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
10、 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
11、 does not bind mouse IgA, IgM or serum albumin. GFP：绿色荧光蛋白（GFP; 26.9 kDa）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
12、 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 orde
13、r of 10-15 mol/L, ranking among one of the strongest known non-covalent interactions.Streptavidin is much less soluble in water than avidin, and it lacks avidins extensive glycosylation. Streptavidin has a mildly acidic isoelectric point (pI) of 5.蛋白标签蛋白标签（proteintag）是指利用 DNA 体外重组技术，与目的蛋白一起融合表达的一种多肽
14、或者蛋白，以便于目的蛋白的表达、检测、示踪和纯化等。随着技术的不断发展，研究人员相继开发出了具有各种不同功能的蛋白标签。目前，这些蛋白标签已在基础研究和商业化产品生产等方面得到了广泛的应用。美国 GeneCopoeia（复能基因）为客户提供 50 多种蛋白标签，可以满足客户的不同需求，包括各种最新型的标签，如：SNAP-Tag、Halo Tag、AviTag、Sumo 等；也提供齐全的各种常用标签，如eGFP、His、Flag 等等标签。以下是部分蛋白标签的特性介绍，更加详细的介绍可在查询克隆产品的结果列表里面看到各种推荐的蛋白标签和载体。 标签 纯化 促进溶解度 抗体效价 细胞标记His6
15、+ +/- +/- Flag + +/- + GST + + + MBP + + + His-MBP + + + HA + eGFP/CFP/YFP +Myc + His-Myc + + His-AviTag + + + Sumo + + His-Sumo + + + SNAP-Tag + + +Halo Tag + + +TrxHISHis6 是指六个组氨酸残基组成的融合标签，可插入在目的蛋白的 C 末端或 N 末端。当某一个标签的使用，一是能构成表位利于纯化和检测；二是构成独特的结构特征（结合配体）利于纯化。组氨酸残基侧链与固态的镍有强烈的吸引力，可用于固定化金属螯合层析(IMAC)，对重
16、组蛋白进行分离纯化。使用 His-tag 有下面优点： 标签的分子量小，只有0.84KD，而 GST 和蛋白 A 分别为26KD 和30KD ，一般不影响目标蛋白的功能； His 标签融合蛋白可以在非离子型表面活性剂存在的条件下或变性条件下纯化，前者在纯化疏水性强的蛋白得到应用，后者在纯化包涵体蛋白时特别有用，用高浓度的变性剂溶解后通过金属螯和亲和层析去除杂蛋白，使复性不受其它蛋白的干扰，或进行金属螯和亲和层析复性； His 标签融合蛋白也被用于蛋白质- 蛋白质、蛋白质-DNA 相互作用研究； His 标签免疫原性相对较低，可将纯化的蛋白直接注射动物进行免疫并制备抗体。 可应用于多种表达系统，纯化的条件温和； 可以和其它的亲和标签一起构建双亲和标签。 Flag 标签蛋白Flag 标签蛋白为编码 8 个氨基酸的亲水性多肽（DYKDDDDK），同时载体中构建的 Kozak 序列使得带有FLAG 的融合蛋白在真核
