tailieunhanh - Báo cáo khoa học: Gc recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions

Weak and transient protein–protein interactions are associated with biolog-ical processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein–protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. | IFEBS Journal Gy recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions Nobuo Fukuda1 Jun Ishii2 and Akihiko Kondo1 1 Department of ChemicalScience and Engineering Graduate Schoolof Engineering Kobe University Japan 2 Organization of Advanced Science and Technology Kobe University Japan Keywords Gy recruitment system G-protein signal mating transient protein-protein interactions yeast Correspondence A. Kondo Department of ChemicalScience and Engineering Graduate School of Engineering Kobe University 1-1 Rokkodaicho Nada-ku Kobe 657-8501 Japan Fax 81 78 803 6196 Tel 81 78 803 6196 E-mail akondo@ Received 5 April 2011 revised 20 May 2011 accepted 5 July 2011 doi Weak and transient protein-protein interactions are associated with biological processes but many are still undefined because of the difficulties in their identification. Here we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gy recruitment system which utilizes yeast G-pro-tein signaling and mating growth selection to screen interacting protein pairs. In the current study to test the capability of our method we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gy recruitment system the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity X 103 M-1 was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wildtype haploid cells diploid colonies were vigorously and .

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