Expression in Escherichia coli and purification of human eosinophil-derived neurotoxin with ribonuclease activity.
Eosinophil-derived neurotoxin (EDN) is a ribonuclease with neurotoxic and helminthotoxic properties. It is present in the crystalloid granules of human eosinophils. We report the expression and characterization of a functionally active recombinant human EDN using the pMAL-cRI expression system. A cDNA for mature EDN was obtained by PCR and inserted in pMAL-cRI downstream of the malE gene encoding maltose binding protein. Induction of the ptac promoter of the plasmid in Escherichia coli strain BL21(DE3) resulted in high level expression of soluble MAL-EDN fusion protein. Cleavage of affinity purified fusion protein with Factor Xa protease released recombinant EDN which comigrated with native EDN on SDS-polyacrylamide gels and cross-reacted with a polyclonal anti-EDN antiserum on Western blots. IN contrast to previous attempts at EDN expression, denatured and refolded EDN had ribonuclease activity and was prepared in microgram amounts. The availability of recombinant human EDN should facilitate studies of its structure and biological functions.
Sun
L
L
Department of Cellular Sciences, St. George's Hospital Medical School, University of London, United Kingdom.
Li
M S
MS
Fisher
L M
LM
Spry
C J
CJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D047208:Eosinophil-Derived Neurotoxin; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000378:metabolism / Q000494:pharmacology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009498:Neurotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D033902:Periplasmic Binding Proteins; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012260:Ribonucleases / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009498:Neurotoxins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D007544:Isopropyl Thiogalactoside; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; D015951:Factor Xa