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PEDS Advance Access originally published online on August 3, 2004
Protein Engineering Design and Selection 2004 17(6):501-508; doi:10.1093/protein/gzh060
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Protein Engineering, Design & Selection vol. 17 no. 6 © Oxford University Press 2004; all rights reserved

A system based on specific protein–RNA interactions for analysis of target protein–protein interactions in vitro: successful selection of membrane-bound Bak–Bcl-xL proteins in vitro

Shinya Y. Sawata1, Eigo Suyama1,2 and Kazunari Taira1,2,3

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

3 To whom correspondence should be addressed, at the Tokyo address. E-mail: taira{at}chembio.t.u-tokyo.ac.jp

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 N-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.

Received October 26, 2003; revised April 20, 2004; accepted April 29, 2004.

Edited by Haruki Nakamura


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