Expression of 5 alpha-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5 alpha-reductase.
A cDNA encoding a full-length rat 5 alpha-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5 alpha-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5 alpha-reductase were used as antigens to produce rabbit polyclonal antibodies to 5 alpha-reductase. Antibodies to the 5 alpha-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5 alpha-reductase fusion protein reacted with a single component of rat liver microsomes with M(r) 26,000 on Western blots, consistent with the size of 5 alpha-reductase predicted from its cDNA, and with a M(r) 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M(r) 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5 alpha-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5 alpha-reductase fusion protein antibodies with the trp E-5 alpha-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5 alpha-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissues. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.
Ben May Institute, University of Chicago, IL 60637.
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Hiipakka
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Wang
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Bloss
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Ito
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Liao
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis* / Q000235:genetics; D000818:Animals; D000917:Antibody Formation; D004247:DNA; D004926:Escherichia coli; D005260:Female; D007150:Immunohistochemistry; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D011467:Prostate / Q000201:enzymology; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D011993:Recombinant Fusion Proteins; D004247:DNA; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase