Human beta-defensin-3 and 4 (HBD-3-4) are two low molecular weight cationic peptides with three conserved cysteine disulfide bonds, and exhibit a broad range of antimicrobial activity and do not acquire any microbial resistance. In order to produce these uneasily detectable, degradable and toxic polypeptide efficiently, an alternative approach based on the Escherichia coli cell-free biosynthesis system was proposed. The polypeptide of interest is synthesized as a fusion protein linked to trxA or green fluorescent protein (GFP) through a cleavable spacer. With batch mode operation, significant amount of hBD3-4 fused with trxA or GFP can be expressed in this cell-free system, and the product is soluble and stable. Furthermore, the GFP moiety provides directly visuable and quantitative monitoring of the polypeptide synthesis. This work will be helpful to rapid and visuable expression of other similar defensins using in vitro cell-free system. (c) 2006 Published by Elsevier Ltd.