Intracellular antibodies for proteomics.
Intracellular antibodies for proteomics.
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DOI：
10.1016/j.jim.2004.04.014
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发表时间：
2004-07
影响因子：
2.2
通讯作者：
Cattaneo A
中科院分区：
文献类型：
--
作者：
Visintin M;Meli GA;Cannistraci I;Cattaneo A
细胞内抗体技术在蛋白质组学研究中有着广泛的应用。通过开发新的实验策略，允许在细胞内表达的条件下选择抗体，细胞内抗体用于蛋白质组的系统研究的潜力已经成为可能。细胞内抗体捕获技术（IACT）是一种基于体内双杂交的方法，最初开发用于选择易于折叠以进行异位表达的抗体。IACT已用于快速有效地鉴定细胞内区室中的新型抗原-抗体对，以及用于体内鉴定由选定的细胞内抗体识别的表位。IACT为胞内抗体技术在蛋白质组学中的大规模应用开辟了道路。然而，在其目前的形式中，其用途受到如下限制：需要预先选择蛋白抗原上的输入噬菌体抗体文库，或者需要从针对靶蛋白免疫的小鼠构建抗体文库，以提供富集的输入文库来补偿酵母细胞转化的次优效率。这些富集步骤需要表达相应的蛋白质，这代表了该技术规模扩大的严重瓶颈。我们在此描述了胞内抗体的单罐文库（SPLINT）的构建，该单罐文库是以使得抗原特异性胞内抗体可以直接从基因序列分离的形式直接在酵母细胞质中表达的scFv片段的幼稚文库，而不需要对相应蛋白质进行任何操作。我们还描述了从SPLINT中分离出一组针对许多不同蛋白质的胞内抗体。SPLINT在全基因组范围内的应用将有助于细胞蛋白质组功能组织的系统研究。
The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.
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影响因子：
30.8
作者：
Martienssen, RA
通讯作者：
Martienssen, RA
影响因子：
11.4
作者：
MHASHILKAR, AM;BAGLEY, J;MARASCO, WA
通讯作者：
MARASCO, WA
影响因子：
16.2
作者：
PRIOR, P;SCHMITT, B;BETZ, H
通讯作者：
BETZ, H
影响因子：
2
作者：
PERELSON, AS;OSTER, GF
通讯作者：
OSTER, GF
影响因子：
6.4
作者：
Arafat, W;Gómez-Navarro, J;Curiel, DT
通讯作者：
Curiel, DT