The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety.
We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.
Castro
M G
MG
Department of Biochemistry & Physiology, School of Animal and Microbial Sciences, University of Reading.
Lowenstein
P R
PR
Saphier
P W
PW
Linton
E A
EA
Lowry
P J
PJ
D000818:Animals; D000917:Antibody Formation; D002417:Cattle; D003346:Corticotropin-Releasing Hormone / Q000096:biosynthesis* / Q000276:immunology; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005260:Female; D006241:Haptens / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry; D007124:Immunoenzyme Techniques; D008297:Male; D010902:Pituitary Gland / Q000737:chemistry; D011498:Protein Precursors / Q000096:biosynthesis* / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D012756:Sheep; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000276:immunology
D006241:Haptens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C041645:pro-corticotropin releasing hormone; D003346:Corticotropin-Releasing Hormone; D001616:beta-Galactosidase