tailieunhanh - Báo cáo khoa học: The competitor-introduced Gc recruitment system, a new approach for screening affinity-enhanced proteins

We have developed a new approach based on the Gcrecruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Gcrecruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that mem-brane localization of the G-proteincsubunit (Gc) is essential for signal transduction in yeast. | ễFEBS Journal The competitor-introduced Gc recruitment system a new approach for screening affinity-enhanced proteins Nobuo Fukuda1 Jun Ishii2 Tsutomu Tanaka2 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 affinity enhancement competitor-introduced system directed evolution G-protein signaling yeast two-hybrid 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 8 December 2009 revised 18 January 2010 accepted 26 January 2010 doi We have developed a new approach based on the Gy recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Gy recruitment system is a yeast two-hybrid Y2H system that utilizes G-protein signaling and is based on the fact that membrane localization of the G-protein y subunit Gy is essential for signal transduction in yeast. In the original Y2H system an engineered Gy that lacks membrane localization upon deletion of the lipid modification site Gycyto is produced and a candidate protein with an artificial lipidation site and its counterpart fused with Gycyto are expressed. As protein-protein interactions bring Gycyto towards the plasma membrane G-protein signaling can be activated and the interaction is detected by various cellular responses as the readout. In the current study we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counter-part-Gycyto fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z