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.
Nakajima
M
M
Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Hirakata
M
M
Nittoh
T
T
Ishihara
K
K
Ohuchi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000096:biosynthesis; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis; D012260:Ribonucleases / Q000378:metabolism; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development
D001798:Blood Proteins; D018076:DNA, Complementary; D047091:Eosinophil Granule Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D012260:Ribonucleases