by on The IBPC is operating a screening and production platform for the cell-free (CF) synthesis of membrane proteins and for the labeling of proteins with stable isotopes in E. coli lysates. The platform includes the in-house preparation of CF lysates tuned for either high yield (HY-S30), high folding capacity (HS-S30) or reduced label background (Stablelabel-S30)). Furthermore, our group prepares libraries of preformed nanoparticles containing defined membrane compositions.
Our stepwise process for CF protocol development includes (i) the systematic screening and optimization of protein yield in the P-CF expression mode and (ii) the analysis of cotranslational membrane protein solubilization in defined lipid or detergent environments in the L-CF or D-CF modes.5-7 In addition, the system can be adjusted to suit individual targets by supplying stabilizing ligands, interaction partners or other compounds (Fig. 1).
Fig. 1. Cotranslational insertion of a CF synthesized GPCR into preformed nanodisc membranes. Exemplified is the production of the human beta-1 adrenergic receptor. Variable compounds of the system are indicated and can be adjusted according to individual targets.
Our target pipeline includes G protein-coupled receptors, membrane integrated enzymes, ion channels and prokaryotic membrane proteins. Final samples suitable for functional or structural analysis can be generated within two days (Fig. 2).
The recently developed nanotransfer technique allows the subsequent simultaneous analysis of CF generated membrane protein samples in vitro and in vivo (Fig. 3). The nanotransfer is a unique and fast method to monitor the function of CF synthesized membrane proteins in various cultivated cell lines or even in primary cells.
Fig. 3. Nanotransfer of CF synthesized H2R into HEK293 cells. The CF synthesized and transferred H2R (blue) dimerizes with cell-based synthesized H2R and H1R GPCRs (green).
