Production of rat renin fusion protein in Escherichia coli and the preparation of renin-specific antisera.
Rat renin fused at the N-terminus with Sj26, a 26,000 Da glutathione S-transferase of Schistosoma japonicum, was expressed in Escherichia coli. The fusion protein was soluble and easily purified from crude bacterial lysates by affinity chromatography on immobilised glutathione. The fusion protein possessed no detectable renin activity. Antisera raised in rabbits against the fusion protein were specific for renin. These antisera did not bind soluble renin but bound immobilized renin. By immunoblotting, these antisera demonstrated rat renin to migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two broad bands of 33,000-34,000 and 35,000-37,000 Da. By immunocytochemistry of rat tissues, these antisera stained renin containing cells in the afferent arteriole of the glomerulus of the kidney, the zona glomerulosa of the adrenal and the corpus luteum of the ovary. However, apart from the afferent arteriole of the kidney, no immunoreactive renin was identified in blood vessels of the kidney, adrenal or ovary. These studies demonstrate that a recombinant renin fusion protein is a valuable alternative approach for the preparation of renin-specific antisera.
Campbell
D J
DJ
St. Vincent's Institute of Medical Research, Fitzroy, Australia.
Valentijn
A J
AJ
Berka
J L
JL
D000818:Animals; D001160:Arterioles / Q000737:chemistry; D002846:Chromatography, Affinity; D003338:Corpus Luteum / Q000737:chemistry; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics; D007106:Immune Sera; D007678:Kidney Glomerulus / Q000098:blood supply; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*; D012083:Renin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D012549:Schistosoma japonicum / Q000235:genetics; D015384:Zona Glomerulosa / Q000737:chemistry
D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase; D012083:Renin