Biotechnology and Bioengineering
Biotechnology and Bioengineering
5 years ago

Development of aptamers against unpurified proteins

Development of aptamers against unpurified proteins
Kaori Tsukakoshi, Kazunori Ikebukuro, Shinichi Goto
SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples. There is growing demand for the development of advanced SELEX techniques not relying on complicated processes for protein purification from native samples or recombinant proteins. In this review, Goto and coworkers summarized several methods, such as Cell-SELEX, In vivo SELEX, subtractive-EMSA SELEX, Two-dimensional-based electrophoresis SELEX (2DE-SELEX), that can directly develop an aptamer from various unpurified sources such as whole cells, tissues, serum, and cell lysates.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26389

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