Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Oct;11(10):1758-65.
doi: 10.1101/gr.180101.

Protein-protein interaction panel using mouse full-length cDNAs

H Suzuki  1 , Y Fukunishi, I Kagawa, R Saito, H Oda, T Endo, S Kondo, H Bono, Y Okazaki, Y Hayashizaki
Affiliations

Protein-protein interaction panel using mouse full-length cDNAs

H Suzuki et al. Genome Res. 2001 Oct.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Strategy for the high-throughput in vivo assay. (A) Design of the gene-specific forward primers. Each gene-specific forward primer was designed to anneal just downstream of the predicted initiation ATG of the gene. Each gene-specific forward primer has a common sequence that is used as a margin to connect the cDNA with other DNA sequences. The common sequence consists of the Shine-Dalgarno (SD) sequence for a prokaryotic ribosome-binding site, GAAGGA, and the Kozak consensus sequence for a eukaryotic translation initiation site, GCCGCCACCATG. (B) Schematic representation of the sample preparation and assay methods. (Thin arrows) PCR primers used; (red boxes) the common sequence region. The assay was performed based on the mammalian two-hybrid system. The pG5luc vector contains five Gal4 binding sites (BD) and a minimal TATA box, both of which are upstream of the luciferase gene; interaction between the BIND and ACT fusion proteins increases luciferase expression. (CDS) Protein coding sequence; (CMV) human cytomegalo virus immediate early promoter; (Gal4) yeast Gal4 DNA-binding domain; (VP16) herpes virus VP16 transcriptional-activation domain. (C) Agarose gel electrophoresis of the PCR-mediated constructs from various lengths of cDNAs. The constructs were prepared by two steps of PCR as described in the Methods. Two microliters of the first PCR products, BIND samples, and ACT samples were subjected to the 1% agarose gel in this order. A mixture of 250 ng of λ-HindIII and 250 ng of φX174-HaeIII was used as the size marker (M). Clone ID of each cDNA and the size of the first PCR product calculated from the nucleotide sequences were as follows: (lanes 1–3) 2010004E10, 0.6 kb; (lanes 4–6) 2310016E22, 1.2 kb; (lanes 7–9) 2310009C19, 1.9 kb; (lanes 10–12) 4931412A05, 3.3 kb. (D) Expression of the fusion proteins from the PCR-mediated samples. The fusion proteins expressed from the BIND samples in C were detected by Western blotting analysis using a monoclonal antibody against the Gal4 DNA-binding domain. Clone ID of each BIND sample and the size of the fusion proteins calculated from the deduced amino acid sequences were as follows: (lane 1) 2010004E10, 30 kD; (lane 2) 2310016E22, 57 kD; (lane 3) 2310009C19, 72 kD; (lane 4) 4931412A05, 101 kD. The size of the fusion protein in lane 4 seemed to be slightly larger than the calculated size. It is unclear whether it may be because of the posttranslational modifications.
Figure 2
Figure 2
Flow chart of the high-throughput assay system. (BKG, background).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al. The genome sequence of Drosophila melanogaster. Science. 2000;287:2185–2195. - PubMed
    1. Cochran AG. Antagonists of protein–protein interactions. Chem Biol. 2000;7:R85–R94. - PubMed
    1. Colas P, Brent R. The impact of two-hybrid and related methods on biotechnology. Trends Biotechnol. 1998;16:355–363. - PubMed
    1. Dang CV, Barrett J, Villa-Garcia M, Resar LM, Kato GJ, Fearon ER. Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization. Mol Cell Biol. 1991;11:954–962. - PMC - PubMed
    1. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci. 1998;95:14863–14868. - PMC - PubMed

Publication types