Abstract
Asthma is an increasing problem in industrial countries. Eosinophil cationic protein (ECP) has been suggested as a factor in allergic respiratory diseases; it is also a biomarker for detecting the severity of asthma. ECP, a peptide of 160 amino acids, is secreted by activated human eosinophils and is characterized as a cytotoxin against bacteria, parasites, and epithelial cells in the trachea. Sequence homology among ECP and other human ribonucleases revealed that ECP belongs to the RNase A family. Of the RNase A family, eosinophil-derived neurotoxin (EDN) is most similar to ECP. Recombinant proteins with six histidines behind the C-terminus of ECP and EDN were designed and expressed in E. coli. The mature recombinant ECP and EDN were obtained from pET3a-mecp-6H/BL21 (DE3) and pRSETA-medn-6H/BL21 (DE3), respectively. The recombinant proteins formed inclusion bodies inside E. coli cells. They were purified by a His-tag chromatography column and refolded in a solution containing glutathion (oxidized/reduced form). The RNase activity monitored by RNA-casting gel showed that the refolded ECP recovered the ability to digest RNA. But in the hemolysis assay, the refolded ECP could not damage the red blood cells as the mature ECP had. Chimeric ECP/EDN constructs were ligated into pRSETA and transformed into E. coli C43 (DE3). The chimeric proteins probed by anti-EDN monoclonal antibody showed that EDN and chimeric A could be recognized. It indicated that the anti-EDN mAb recognize the P85-P117 region of EDN. The largest difference between ECP and EDN was in this region. Therefore, P85-P117 may be the key region for distinguishing the properties of ECP and EDN.