Adenine nucleotide translocator is an important member of the mitochondrial carrier family, which is involved in the transportation of various metabolites. In the present work, the Escherichia coli cell-free system was chosen as an alternative way to express this highly toxic membrane protein. The expression level of insoluble sjANT from Schistosoma japonicum in E. coli cell-free system could attain 472 mu g/mL, about 25.2 times improvement over the previous report. The obtained insoluble sjANT can be resolubilized with different detergents. Among them, Digitonin could effectively solubilize approximate 38% of the target membrane protein. Moreover, sjANT can be further expressed in the hydrophobic E. coli cell-free system as a soluble form with presence of different detergents. The results suggested that Digitonin and Brij 58 were two ideal candidates to support high expression of soluble sjANT, and the highest soluble expression level (182 mu g/mL) was achieved with the supplementation of 0.4% (v/v) Digitonin in the cell-free system. The present work has provided a rapid and efficient procedure to express the complex and highly toxic membrane protein in the cell-free system, and will be beneficial to construct a novel drug-discovery model to screen the sjANT-based inhibitors for schistosomiasis treatment. (C) 2011 Elsevier Ltd. All rights reserved.