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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.

Background: Hepatitis B virus (HBV) infection is a serious health problem worldwide. Treatment recommendation and response are mainly indicated by viral load, e antigen (HBeAg) seroconversion, and ALT levels. The S antigen (HBsAg) seroconversion is much less frequent. Since HBeAg can be negative in the presence of high viral replication, preS antigen (HBpreSAg) might be a useful indicator in management of chronic HBV infection.Results: A new assay of double antibody sandwich ELISA was established to detect preS antigens. Sera of 104 HBeAg-negative and 50 HBeAg-positive chronic hepatitis B patients have been studied and 23 HBeAg-positive patients were enrolled in a treatment follow-up study. 70% of the HbeAgpositive patients and 47% of the HBeAg-negative patients showed HBpreSAg positive. Particularly, in the HBeAg-negative patients, 30 out of 47 HBpreSAg positive patients showed no evidence of viral replication based on HBV DNA copies. A comparison with HBV DNA copies demonstrated that the overall accuracy of the HBpreSAg test could reach 72% for active HBV replication. HBpreSAg changes were well correlated with changes of HBsAg, HBV DNA and ALT levels during the course of IFN-α treatment and follow-up. HBeAg positive patients responded well to treatment when reduction of HBpreSAg levels was more pronounced.Conclusion: Our results suggested that HBpreSAg could be detected effectively, and well correlated with HBsAg and HBV DNA copies. The reduction of HBpreSAg levels in conjunction with the HBV DNA copies appears to be an improved predictor of treatment outcome.