CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was puriWed to near homogeneity by a two-step FPLC procedure, Wrst by Ni2+ aYnity chromatography followed by a gel-Wltration chromatography, which yielded about 10mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-Wltration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulWde bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulWde bonds and to stabilize the protein. Successfully growing crystals of the puriWed CARP protein also proved that we can produce well folded CARP protein in E. coli.