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, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). In rats, 8 homologues of human ECP and EDN have been identified. To clarify the biological activity of rat eosinophil ribonucleases, we cloned rat eosinophil-associated ribonuclease (EAR)-1/rat ribonuclease 7 and rat EAR-2/rat ribonuclease 4, and produced recombinant rat pre-EAR-1 and pre-EAR-2 in a bacterial expression system. METHODS: As we have already cloned the complete nucleotide 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 pre-EAR-2 were expressed in Escherichia coli as N-terminal 6 x histidine-tagged proteins, isolated from the insoluble fraction of the cell lysate and purified by a single-step method using an Ni-NTA resin column after solubilization with a 6 M guanidine solution. RESULTS: The deduced amino acid sequence revealed that the molecular weight of EAR-2 containing the signal peptide 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 Staphylococcus aureus. CONCLUSIONS: These findings suggest that rat EAR-1 and EAR-2 act as host defense factors against bacterial infection in rats.[1]References
- Expression and purification of recombinant rat eosinophil-associated ribonucleases, homologues of human eosinophil cationic protein and eosinophil-derived neurotoxin, and their characterization. Nakajima, M., Hirakata, M., Nittoh, T., Ishihara, K., Ohuchi, K. Int. Arch. Allergy Immunol. (2001) [Pubmed]
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