Cell-free production of biologically active polypeptides: Application to the synthesis of antibacterial peptide cecropin

An approach to preparative production of polypeptides, including uneasily t estable, degradable, and toxic ones, is proposed on the basis of in vitro e xpression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The appro ach implies that a polypeptide of interest is synthesized as a fusion prote in with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantit ative monitoring of the polypeptide synthesis, as well as solubility and st ability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated. (C) 2001 Academic Press.