The cell-free protein synthesis system, which is sometimes called in vitro expression, has been remarkably improved in the past decade. However, proteins containing disulfide bonds could not been produced as active forms using the conventional cell-free system because it was carried out under reduced conditions. We have demonstrated that modification of the reaction conditions, i.e., no addition of reducing reagent, removal of the reducing reagent from the E coli S30 extract by dialysis, and the addition of some molecular chaperons, enabled the correct folding of such proteins as Burkholderia cepacia lipase, Streptomyces antibioticus phospholipase D, Phanerochaete chrysosporium manganese peroxide (MnP), and artificial antibodies (scFv and Fab). As an application of cell-free protein synthesis, we have developed a novel high-throughput protein library construction system by combining the amplification of a single DNA molecule by means of PCR with a cell-free protein expression system. This completely cell-free system is termed SIMPLEX (single-molecule-PCR-linked in vitro expression). By applying SIMPLEX, we have achieved (1) inversion of the enatioselectivity of the bacterial lipase, (2) improvement in the H2O2 stability of the fungal MnP, and (3) enhancement of the binding affinity of anti-human-serum-albumin scFv.