High-yield cell-free protein production from P-gel

Cell-free systems represent a promising approach to quickly and easily produce preparative amounts of proteins. However, it is still challenging to obtain high volumetric yields (>mg ml(-1)) of proteins from the present cell-free systems. This protocol presents a cell-free protein synthesis method using a novel DNA gel that dramatically increases protein yield compared with current systems. This protein-producing gel (termed 'P-gel system' or 'P-gel'), which consists of genes as part of the gel scaffolding, can produce mg ml(-1) amounts of functional proteins. This protocol describes steps pertaining to plasmid design, fabrication of P-gel molds, formation of P-gel micropads and cell-free protein expression with an expected yield of up to 5 mg ml(-1) of functional Renilla luciferase (Rluc). This entire process can take 1-3 d, depending on the desired quantity of protein.