Protein–Protein Interaction Panel Using Mouse Full-Length cDNAs (original) (raw)

  1. Harukazu Suzuki1,
  2. Yoshifumi Fukunishi1,
  3. Ikuko Kagawa1,
  4. Rintaro Saito1,
  5. Hiroshi Oda1,
  6. Toshinori Endo1,
  7. Shinji Kondo1,
  8. Hidemasa Bono1,
  9. Yasushi Okazaki1, and
  10. Yoshihide Hayashizaki1,3
  11. 1Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan; Genome Science Laboratory, RIKEN Tsukuba Institute, Tsukuba 305-0074, Japan; 2Department of Medicine, Tsukuba University, Tsukuba 305-0006, Japan.

Abstract

We have developed a novel assay system for systematic analysis of protein–protein interactions (PPIs) that is characteristic of a PCR-mediated rapid sample preparation and a high-throughput assay system based on the mammalian two-hybrid method. Using gene-specific primers, we successfully constructed the assay samples by two rounds of PCR with up to 3.6 kb from the first-round PCR fragments. In the assay system, we designed all the steps to be performed by adding only samples, reagents, and cells into 384-well assay plates using two types of semiautomatic multiple dispensers. The system enabled us examine more than 20,000 assay wells per day. We detected 145 interactions in our pilot study using 3500 samples derived from mouse full-length enriched cDNAs. Analysis of the interaction data showed both several significant interaction clusters and predicted functions of a few uncharacterized proteins. In combination with our comprehensive mouse full-length cDNA clone bank covering a large part of the whole genes, our high-throughput assay system will discover many interactions to facilitate understanding of the function of uncharacterized proteins and the molecular mechanism of crucial biological processes, and also enable completion of a rough draft of the entire PPI panel in certain cell types or tissues of mouse within a short time.

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