Cell-free expression system produces proteins in vitro through translation machineries extracted from cells. It has become a well-developed technique for synthesizing proteins in cell lysates. This approach meets with the increasing demands for a more straightforward, rapid and efficient way of protein production. Cell-free expression overcomes many obstacles of conventional cellular expression. Firstly, it is ideal for high-throughput screening of mutagenesis. Moreover, the system works independently and is free of cellular context. The whole machinery sets up a better environment to manufacture toxic proteins, membrane proteins and proteins that would be easily degraded by intracellular proteases. In addition, the reaction steps can be simply manipulated by adding/removing certain factors, such as cofactors, energy sources, detergents and so on.