Synthesis of Nanodisc-Stabilized Membrane Protein Antigens Using a Cell-Free System

Take a bacterial cell-free system that lacks intact cellular structures but retains the molecular components necessary for in vitro transcription and translation.

Introduce plasmids encoding the membrane protein antigen and the scaffold protein, along with synthetic phospholipids and polymers.

Supplement the mixture with ATP, GTP, and an amino acid mix to initiate protein synthesis.

Simultaneously, prepare a feed solution enriched with the amino acid mix.

Load the mixture into the inner chamber and the feed solution into the outer chamber of a dual-compartment dialysis device.

During incubation, the substrates diffuse from the outer to the inner chamber through the semi-permeable membrane, facilitating plasmid transcription and protein translation.

The scaffold protein assembles the phospholipids and polymers into lipid nanodiscs and facilitates the folding and incorporation of MPA into these structures.

By-products generated during the reaction diffuse into the outer chamber, maintaining optimal conditions for nanodisc-stabilized MPA formation.

Prepare MOMP-tNLPs by employing a cell-free method.

Defrost the reconstitution buffer from the cell-free protein expression kit two hours before se