Nanodelivery: a novel approach to transfer cell-free expressed GPCRs into live cells
Authors:   , Umbach Simon 
Published: December 2024

In biochemistry and cell biology, DNA/RNA transfection is one of the most fundamental and essential methods for modifying cellular phenotypes. This technique is critically important because it enables the study of protein function and regulation in a distinct cellular environment.
Despite their significance, conventional transfection methods however exhibit significant technical constraints or infeasibility. For example, transfection can occasionally produce inactive proteins, display cytotoxicity and most-importantly is not applicable to all targets since some cell-types are hard or even impossible to transfect.
Recently, a method to transfer membrane proteins (MPs) into live cells, has been described, effectively enabling the modification of cellular phenotypes without any genetical alteration. This approach, dubbed nanotransfer or nanodelivery, relies on the application of nanodiscs (NDs), a MP-solubilizing lipid bilayer that is kept intact by a supporting belt of so-called membrane scaffold proteins (MSP). MP/ND complexes are incubated with cells which results in the incorporation of the ND cargo into the cellular plasma membrane.
The aim of this thesis was to thoroughly evaluate this novel nanodelivery approach, to systematically screen the fundamental parameters and mechanistic details of this method and finally to establish and optimize novel cellular assays with nanotransferred cells. The main targets being a variety of G-protein coupled receptors (GPCRs). GPCRs are integral MPs with seven membrane-spanning helices and, while being one of the most relevant pharmacological targets, are notoriously difficult to express heterologously in cells – making them a perfect target for nanotransfer approaches.
In the last years, cell-free expression of GPCRs has emerged as a viable alternative to conventional cell-based expression systems. Here, the complete transcription and translation machinery is extracted from E. coli and can then be supplied with a DNA/RNA template of choice. The CF system can be optimized and adapted by addition of an energy regeneration system, customized redox conditions and the addition of NDs for the effective solubilization of MPs. In this configuration, GPCR yield of up to 1 mg/ml can be achieved.
The chosen targets of this thesis were a variety of GPCRs ranging from well-studied species such as the endothelin receptor B (ETB) via the free fatty acid receptors FFAR1, FFAR2 and FFAR3 or the histamine receptors H1R and H2R, to the prostacyclin receptor IP and the orphan receptor GPRC5B.
First, CF expression quality and quantity were optimized. Therefore, GPCR solubilization was screened for the optimal ND lipid content, using mNeonGreen (mNG) derivates and size-exclusion chromatography (SEC) profiling. All tested receptors showed strong preference for either DOPG or POPG lipids. Expression yields of the receptors ranged from 0.7 mg/ml for the IP receptor to 1.2 mg/ml for FFAR2. Using FFAR1 as exemplary target, the effects of ND concentration (30, 60 or 90 µM) as well as plasmid concentration (5, 10 or 15 ng/µl) were screened. An inverse correlation was observed, where higher ND concentrations or lowered plasmid concentrations both improved the receptor folding, as assayed via SEC profiling. Then, glutathione- and DTT-based redox systems were compared. For all receptors except IP, the glutathione-based system could improve GPCR/ND particle quality.
By further exploration of a newly established optimization strategy, ligands or cognate mini-G proteins, a miniaturized version of the full-length G proteins, were supplied to the CF reaction.
While tested ligands could effectively improve GPCR folding, only FFAR1 showed significantly improved expression quality. Another recent development in the nanoparticle field was the implementation of circularized NDs (cNDs). These have however never been applied to the CF system. In a first attempt to assay cNDs for their suitability in CF GPCR expression, FFAR1, FFAR2 and FFAR3 were expressed into these novel ND types. While yields were overall similar, SEC profiling revealed massive improvements in receptor folding quality.
Even though GPCR expression quality and quantity could be improved. GPCR/ND particles still displayed significant fractions containing soluble aggregates and/or misfolded receptor. Therefore, more sophisticated purification strategies were explored. For ETB, ligand affinity chromatography (LAC) and for the H2R G protein affinity chromatography (GPAC) were established. By immobilizing the ligand or the cognate mini-G protein, only the active receptor fraction could be isolated from the CF reaction mixture, as seen in massively improved SEC profiles.