Construction of polymerome-based artificial cell membrane

Membrane Proteins (MPs) are key players in many different biological networks, such as signal transduction cascades, cell-cell communication and environment sensing.The structural complexity and range of vital biological functions make them not only of scientific interest but also important targets for therapeutic intervention.However, they are more difficult to work with as compared to soluble proteins, due to their rapid disintegration once they are removed from the embedding plasma membrane.Traditional methods of producing MPs, which include heterologous overexpression cells, extraction from cell membranes, purification, and refolding efforts to preserve native structures are labour intensive and the use of detergents during the extraction and purification may cause protein denaturation.Since 1988, when Zozulia SA et al. first demonstrated the in vitro translation of visual rhodopsin (an important class of MP), cell free protein synthesis (CFPS) has been an evolving tool for MP research.Different from in vivo protein production in host cells, CFPS uses cell extract containing translational machinery (e.g.ribosomes, translation factors and metabolic enzymes) for protein expression.The absence of cell membrane creates an open and versatile environment that can be optimized exclusively towards quality production of a single protein product without supporting cell viability and growth.Such characteristics allow maximum modification and control of expression/isolation conditions to overcome issues in conventional in vivo expression systems (e.g.no expression or cell death due to target toxicity, protein denaturation in extraction), making CFPS a valuable alternative tool for challenging protein target production, e.g.MPs.Furthermore, it allows modifying the components that are amenable for expressing membrane proteins (e.g., additional chaperons to assist membrane proteins folding) with flexible strategies as well as high throughput screening when necessary.In the research involving MPs, however, CFPS are facing similar challenges as in traditional methods such as the expressed MPs have to be stabilized and maintain structural Abstract ii integrity during expression and processing.MPs are intrinsically unstable and in in vivo synthesis, the current approaches depend largely on trial-and-errors (e.g.expression system optimization, directed evolution, condition screening).In CFPS, such challenges are approached by supplying amphiphilic components, e.g.membrane support like liposomes (lipid-based vesicles), to mimic cellular environment and to assist MP folding and stabilization.However, a key disadvantage of conventional membrane supports is their limited repertoire of physicochemical properties.Consequently, their applications in CFPS are restricted to genetically stabilized, well-studied targets (e.g.β2AR, AqpZ, bacteria rhodopsin) whereas the majority of challenging MPs (e.g.class B GPCRs) with wild-type sequences have not been attempted.In order to overcome these challenges, more flexible and robust biomimetic membrane systems are highly desired.In an entirely different field, self-assembled block copolymer vesicles (polymersomes) have emerged as suitable candidates in addition to lipid membranes.The chemical diversity allows tuning of membrane properties (e.g.chain length, hydrophobicity, rigidity) over a wider range than traditional membrane supports.They have been explored and show great potential as artificial cell membranes.In this work, we systematically investigated polymersomes as native-like membrane support for MP production in CFPS.Following previous proof of concept studies, we have selected Class B GPCRs, which are important drug targets in many human diseases, including diabetes, osteoporosis and obesity.In comparison to Class A GPCRs, they are more inherently unstable and lack high-affinity ligands for stabilization thus the study of this class of GPCRs has been considerably delayed.Specifically, a type II diabetes target, the Glucagon-like peptide-1 receptor (GLP-1R) was selected because of its role in chronic metabolic disorder and the very limited information available on the characterization of this protein target.Prevalence of type II diabetes has been increasing steadily globally yet no cure is available.As a diabetes target, GLP-1R related treatments offer unique benefits that include not only control of blood glucose level but improvement of co-morbid conditions.