Expression and purification of recombinant rat eosinophil-associated ribonucleases, homologues of human eosinophil cationic protein and eosinophil-derived neurotoxin, and their characterization

Background. Human eosinophils contain two eosinophil ribonucleases, eosinop hil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats , 8 homologues of human ECP and EDN have been identified. To clarify the bi ological activity of rat eosinophil ribonucleases, we cloned rat eosinophil -associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribon uclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacter ial expression system. Methods:As we have already cloned the complete nucle otide sequence for rat EAR-1, we determined that for rat EAR-2 cDNA by the rapid amplification of cDNA ends procedure. Recombinant rat pre-EAR-1 and p re-EAR-2 were expressed in Escherichia coli as N-terminal 6 x histidine tag ged proteins, isolated from the insoluble fraction of the cell lysate and p urified by a single-step method using an Ni-NTA resin column after solubili zation with a 6 M guanidine solution. Results: The deduced amino acid seque nce revealed that the molecular weight of EAR-2 containing the signal pepti de is 17.3 kD and the isoelectric point is 8.59. The homology in amino acid sequence between rat pre-EAR-2, and human pre-ECP and human pre-EDN is 51 and 53%, respectively. The purified and refolded recombinant rat pre-EAR-1 and pre-EAR-2 showed bactericidal activity against E. coli and Staphylococc us aureus. Conclusions: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats. Copyright (C) 2001 S. Karger AG, Basel.