br Materials and methods br Results discussion
Materials and methods
Results + discussion
Conclusions We have demonstrated the systematic optimization of the functional folding of a CF synthesized GPCR. The approach addresses central issues relevant for many membrane proteins such as disulfide bridge formation, proper hydrophobic environments or chaperone dependent folding. The key findings can thus be applied as blueprint for the quality optimization of other membrane proteins. The exemplified ETB receptor is representative for the most abundant rhodopsin class of GPCRs, being in focus of pharmaceutical and medical research. The obtained productivity of 1.6 nmol CI 976 australia out of a single mL of CF reaction is already highly competitive if compared to the conventional expression systems based on cell cultures. The thermostabilized ETB synthesized in E. coli cells yielded 0.6–2.4 pmol per mg total membrane protein, whereas protein synthesis in Sf9 cells amounted to some 80 pmol per mg total membrane protein . Significant amounts of 800 pmol binding sites per mL CF reaction were already obtained with the non-engineered wild type receptor. The established CF platform is therefore interesting for the characterization of full-length GPCRs in native-like membrane environments. The obtained GPCR yield is sufficient for numerous applications such as drug screening or biochemical studies even in throughput formats. In addition to the high productivity, the CF protein synthesis approach takes less than 24 h for obtaining the purified GPCR inserted into defined lipid environments. The small volumes and open accessible reaction further facilitate otherwise cost-intensive modifications such as GPCR labelling and protein synthesis in presence of expensive ligands or modifying enzymes such as lipid- or glycosyl transferases. The majority of the synthesized ETB receptor still remains non-functional in our assay, indicating further significant potential for quality optimization. To our opinion, proper full insertion of ETB into the empty ND membranes might represent a major bottleneck for its folding process. We speculate that the major fraction of solubilized ETB is only partially inserted into the ND membranes and thus not ligand binding competent. The co-translational and translocon independent insertion of membrane proteins into empty ND membranes is a new and artificial approach underlying yet unknown mechanisms. Topology of individual NDs may play a role and not all NDs might be equally insertion competent, as indicated by the observed improvement of ETB folding by ND overtitration. Kinetics of translation and efficient initial interaction of nascent polypeptides with the NDs might play further roles. We could recently demonstrate that membrane protein insertion into preformed NDs shows some cooperative effects and results into the release of a corresponding amount of lipids from the membrane . Revealing the molecular mechanisms and relevant factors for this artificial process will most likely further impact the efficiency of GPCR and other membrane protein production by cell-free synthetic biology.
Acknowledgements We thank S. James Remington (Department of Physics and Institute of Molecular Biology, University of Oregon, USA) for providing the roGFP1-iE construct. This work was funded by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG).