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PEDS Advance Access published online on August 3, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh060
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Received October 26, 2003
Revised April 20, 2004
Accepted April 29, 2004

Article

A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: Successful selection of membranebound Bak-Bcl-xL proteins in vitro

Shinya Y. Sawata 1, Eigo Suyama 2, Kazunari Taira 2*

1 Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan
2 Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-8656, Japan

* To whom correspondence should be addressed. E-mail: taira{at}chembio.t.u-tokyo.ac.jp.


  Abstract

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the amino terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.

Keywords: in vitro selection; RNA-protein interaction; MS2 coat protein; ribosome display; atomic force microscopy.
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