SNAP-Tag-Reactive Lipid Anchors Enable Targeted and Spatiotemporally Controlled Localization of Proteins to Phospholipid Membranes

Abstract

The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. Here we report a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O(6)-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. To our knowledge, this is the first use of SNAP-tag reactions to modify benzylguanine-functionalized lipid membranes. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.

A bioinspired and biocompatible ortho-sulfiliminyl phenol synthesis

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Recent Progress in Micro/Nanoreactors toward the Creation of Artificial Organelles

“…Important advances in the development of minimal synthetic cells employing liposomes have been reported by Devaraj and co‐workers . In particular, Rudd et al reported a new method for the membrane localization of proteins making use of benzylguanine‐modified lipid anchors able to covalently attach the mutant O 6 ‐methylguanine‐DNA methyltransferase (SNAP‐tag) and demonstrated that the system was compatible with cell‐free expression. In particular, the authors employed giant unilamellar liposomes composed of dioleoylphosphatidylcholine and 5 mol% of benzylguanine‐modified lipid anchors.…”

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