Controlling Protein Enrichment in Lipid Sponge Phase Droplets using SNAP‐Tag Bioconjugation

Abstract

All cells use organized lipid compartments to facilitate specific biological functions. Membrane‐bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid‐based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)‐modified phospholipids that form stable covalent linkages with an O6‐methylguanine DNA methyltransferase (SNAP‐tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid‐aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP‐tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid‐based artificial organelles.

An Abiotic Phospholipid Metabolic Network Facilitates Membrane Plasticity in Artificial Cells

“…A notable increase in assembly size was observed during the transition from lamellar phase vesicles to nonlamellar lipid sponge phase droplets. From the time-lapse microscopy, we attributed the size increase to fusion events that frequently occurred between individual droplets, similar to what has previously been observed (24,25). This observation prompted us to investigate whether phospholipid metabolism could be utilized to trigger artificial cell fusion events and facilitate the interchange of membrane components between two initially separated lipid assemblies.…”

“…For instance, during germination, etioplasts in plant cells transition from lamellar to nonlamellar membranes upon exposure to light (22,23). Despite previous investigations on the utilization of nonlamellar lipid phases in artificial cells (24)(25)(26)(27)(28)(29)(30)(31), the precise control of phase transitions within these systems remains a significant challenge (32)(33)(34)(35).…”

Coacervate Droplets for Synthetic Cells

“…[58] In another example, benzylguanine-modified phospholipids were incorporated in lipid sponge coacervates and shown to covalently capture SNAP-tag fusion proteins. [130] Other works used nucleotide hybridization in DNA nanostar coacervates to selectively sequester complementary dsDNA [131] or ssDNA [132] strands, as well as protein-DNA conjugates. [133] Excitingly, the use of specific interactions opens the possibility to achieve controllable uptake and release of guest species.…”

Characterizing the Self-Assembly Properties of Monoolein Lipid Isosteres

“…As such, nonlamellar lipid phases have found widespread application in structural biology and biomedicine. , The most common nonlamellar phases are the lipidic cubic, hexagonal, and sponge phases . Nonlamellar lipid mesophases have been exploited as drug delivery systems and food emulsifiers due to their biocompatibility, nontoxicity, and stability in excess water. − They have also been used frequently in structural biology to promote the crystallization of membrane proteins. , Additionally, the ability of some mesophases to encapsulate and recruit hydrophilic or hydrophobic molecules while maintaining their structural integrity has led to their use as nanoreactors for chemical reactions and enzyme immobilization. − …”

“… 19 , 20 Additionally, the ability of some mesophases to encapsulate and recruit hydrophilic or hydrophobic molecules while maintaining their structural integrity has led to their use as nanoreactors for chemical reactions and enzyme immobilization. 21 − 28 …”