Poster: Emerging technologies

Abs # 912: A high throughput in vitro system for analysis of protein-protein interaction

Presenter:	Song, Keming , ksong@sial.com
Authors	Song, Keming  (A)   Uder, Stephanie  (A)   Opper, Kristen  (A)   Juehne, Tom  (A)   Haag, Jeremy  (B)
Affiliations:	(A): Sigma-Aldrich (B): Biology Department, Washington University

Protein-protein interaction (PPI) is involved in almost all fundamental processes in cells. With completion of the human and Arabidopsis genome projects, there has been increasing interest in studying PPI at a genome-wide scale or constructing an “interactome”. However, existing technologies for PPI studies suffer either from a high rate of false positives (e.g. two hybrid systems) or low throughput (e.g. conventional resin-based co-immunoprecipitation method and Biological Resonance Energy Transfer method). In this report, we describe a high throughput in vitro system for PPI analysis. The core of this technology includes a novel ligation-mediated PCR method for rapid construction of expression-ready templates, a coupled one-step transcription/translation system for protein synthesis, and high performance capture plates coated with antibodies that have high affinity to epitope tags. With this system, a target gene can be amplified by standard PCR using a dNTP mix containing thio-dNTPs. The PCR product is then digested with exonuclease III to generate 5’ and 3’ cohesive ends for ligation. Using exonulease III instead of restriction enzymes to create cohesive ends in the first round PCR product is the unique feature of our system since it eliminates the concern of internal restriction sites in the PCR product and the laborious procedure for double restriction enzyme digestion, making this step amenable for high throughput. The PCR product digested with exonuclease III is ligated with a pair of anchors that contain promoter, epitope tag, and terminator sequences. A second round of PCR is then conducted using primers homologous to the anchor sequences to create an expression-ready template (ERT). An ERT usually contains a promoter, an epitope tag, a target gene sequence, and a terminator sequence, and can be directly added to a coupled one-tube transcription/translation reaction (either rabbit reticulocyte or wheat germ system) for synthesis of in vitro epitope-tagged bait/prey proteins. Subsequently, a bait protein and its interactive partners are captured on an antibody-coated 96-well plate via epitope tags. The captured protein complex may be eluted for Western and MALDI analysis or be detected in plate by an ELISA format using target protein-specific or epitope tag-specific antibodies combined with universal detection chemistries. The entire system has been characterized using several PPI models, including human p53/T-antigen, p65/IkB, and Arabidopsis Histone3/Histone4. The results demonstrate that this platform is a rapid, sensitive, and universal tool for PPI study of any organism, and is particularly useful for plant research where there is no efficient system available for protein expression. The technology can be used for confirmation of PPIs generated by two hybrid systems or as a screening tool for PPI studies on a large number of known genes in a matrix approach.