Abstract
Asthma is an increasing problem in industrial countries. Eosinophil cationic protein (ECP) has been suggested as a major causative factor in allergic respiratory diseases, and it is used as a biomarker currently which reflects the severity of asthma. ECP, a single peptide with 160 amino acids, is a toxin secreted by activated human eosinophils. There is a significant sequence homology between ECP and other mammalian ribonuclases. It has anti-parasitic, antibacterial, neurotoxic and ribonuclease activities. ECP also possesses pore-forming activity in damaging the membranes of target cells through transmembrane channels or hydrolysis of the diester-bond of lipids. The ECP reserves the active sites of ribonuclease activivty, His-42, Lys-65, His-155. We are curious about the relationship between the ribonuclease activity and the pore-forming activity of ECP. Here we report the construction and large heterologous expression of recombinant ECP into pET32a/E.coli BL21 (DE3, pLysS) system. The resulted fusion protein is 32 kD and contains thioredoxin at the N-terminus. Purification of overexpressed ECP has been carried out with His-Bind affinity column chromatography, enterokinase digestion, and cation-exchange chrotamography. Unfortunately, there is no haemolysis activity of purified ECP. We also construct recombinant ECP into pRSETA/ E.coli BL21 (DE3, pLysS) as a screening system for cytotoxicity assay of mutant ECP. The results show that first two residues of ribonuclease active site, His42, Lys 65, may not be involved in the cytotoxicity, but His155 might be.