Identification of Protein Interacting Partners Using Tandem Affinity Purification
Identification of Protein Interacting Partners Using Tandem Affinity Purification
复制标题
DOI：
10.3791/3643
复制
发表时间：
2012-02-01
影响因子：
1.2
通讯作者：
Goodfellow, Ian
中科院分区：
文献类型：
--
作者：
Bailey, Dalan;Urena, Luis;Goodfellow, Ian
在理解宿主和病毒蛋白质的功能中，一个关键且通常是限制性的步骤是鉴定相互作用的细胞或病毒蛋白质伴侣。有许多方法可以识别相互作用的伴侣，包括酵母双杂交系统，以及使用重组蛋白和内源性蛋白免疫沉淀的下拉测定，然后进行质谱鉴定（1）。最近的研究强调了双亲和标签介导的纯化的实用性，再加上两个特定的洗脱步骤，在相互作用的蛋白质的鉴定。这种方法，称为串联亲和纯化（TAP），最初用于酵母（2，3），但最近已适用于哺乳动物细胞（4-8）。作为概念验证，我们已经建立了使用充分表征的真核翻译起始因子eIF 4 E的串联亲和纯化（TAP）方法（9，10）。细胞翻译因子eIF 4 E是参与帽依赖性翻译起始的细胞eIF 4F复合物的关键组分（10）。本研究中使用的TAP标签由两个蛋白G单位和一个链霉亲和素结合肽组成，它们被烟草蚀纹病毒（TEV）蛋白酶切割序列分开。本研究中使用的TAP标签由两个蛋白G单元和一个链霉亲和素结合肽组成，它们被烟草蚀纹病毒（TEV）蛋白酶切割序列隔开（8）。为了放弃产生克隆细胞系的需要，我们开发了一种快速系统，该系统依赖于TAP标记的诱饵蛋白从基于pMEP 4（Invitrogen）的附加型维持质粒的表达。使用氯化镉诱导的金属硫蛋白启动子控制来自该质粒的标记的鼠eIF 4 Et 3表达细胞的裂解和随后的亲和纯化（通过结合兔IgG琼脂糖、TEV蛋白酶切割、结合链霉亲和素连接的琼脂糖和随后的生物素洗脱）鉴定了许多明显特异于eIF 4 E下拉的蛋白质（当与仅表达TAP标签的对照细胞系相比时）。通过从1D SDS-PAGE和随后的串联质谱中切除条带来获得蛋白质的身份。鉴定的组分包括已知的eIF 4 E结合蛋白eIF 4G和4 EBP-1。此外，鉴定了eIF 4F复合物的其他组分，即eIF 4A和Poly-A结合蛋白，其中eIF 4 E是eIF 4F复合物的组分。不仅能够识别已知的直接结合伴侣以及二级相互作用蛋白，进一步突出了这种方法在表征未知功能的蛋白质中的实用性。
A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8).As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10). The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.