Modern tools for identification of nucleic acid-binding proteins

Nadia Hégarat^{a, b}, Jean-Christophe François^{a, b,} and Danièle Praseuth^{a, b,}

^aINSERM, U565 Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France

^bMuséum National d'Histoire Naturelle (MNHN) USM 503, CNRS UMR 5153 “Acides Nucleiques: dynamique, ciblage et fonctions biologiques”, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France

Available online 12 April 2008.

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Abstract

Numerous biological mechanisms depend on nucleic acid–protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.

Keywords: Nucleic acid-binding protein; Protein library; Affinity chromatography; Multiprotein complexes

Article Outline

1. Introduction

2. From protein libraries

3. From protein extracts

4. Identification of multiprotein complexes interacting with nucleic acids

5. Conclusions

Acknowledgements

References

Fig. 1. Protein production and immobilization on a chip. (A) A plasmid coding for a tagged protein is immobilized on an array. The microarray is incubated with rabbit reticulocyte lysate to permit fusion protein expression. Tagged protein is immobilized on chip via its interaction with an antibody directed against the tag. (B) Each ORF is cloned in a vector that is suitable for protein expression in the chosen host cell. Proteins are then purified and spotted on slides. In both cases, protein microarrays are incubated with a nucleic acid, labelled with a fluorochrome, for example. After several washes, the interacting proteins are detected using fluorescence. Using the ORF sequence, the identity of the protein is determined.

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