An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins
journal contributionposted on 15.08.2020, 14:03 by Tatsuyuki Yoshii, Kai Tahara, Sachio Suzuki, Yuka Hatano, Keiko Kuwata, Shinya Tsukiji
The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical–genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.
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cell-permeable chemical probeLipidation-Induced Plasma Membrane ...m D cBCPintracellular SNAP-tag lipidation3KPISNAP-tag fusion proteinSNAP-Tag Fusion ProteinsSNAP-tag fusion proteinsK 6-SNAP Δlipidation-induced PMD Cys lipopeptidomimeticSNAP f proteinpost-translational covalent lipopep...intracellular protein targets