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Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

T. S. Grewal, P. J. Lowry, and D. Savva

ABSTRACT

A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

DOI:
https://doi.org/10.1677/jme.0.0030105
Volume 3: Issue 2,
Pages:
105–112 (8 total)
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Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion

M. G. Castro, J. Brooke, A. Bullman, M. Hannah, B. P. Glynn, and P. J. Lowry

ABSTRACT

The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1–76), ACTH, β-lipotrophin and β-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. ArT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1–41). On stimulation with the secretagogue noradrenaline, CRH(1–41) was released into the medium, while the precursor was not. Whilst treatment of wild-type ArT-20 cells with exogenous CRH(1–41) (1 nm) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.

DOI:
https://doi.org/10.1677/jme.0.0070097
Volume 7: Issue 2,
Pages:
97–104 (8 total)
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The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety

M. G. Castro, P. R. Lowenstein, P. W. Saphier, E. A. Linton, and P. J. Lowry

ABSTRACT

We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to β-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 preproCRH 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.

DOI:
https://doi.org/10.1677/jme.0.0050221
Volume 5: Issue 3,
Pages:
221–230 (10 total)
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Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide

S. Jackson, B. A. Spruce, D. M. Glover, B. P. Glynn, and P. J. Lowry

ABSTRACT

Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a β-galactosidase—preproenkephalin A(69– 207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33–5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23·3, 18·2 and 12·6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18·2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.

DOI:
https://doi.org/10.1677/jme.0.0020119
Volume 2: Issue 2,
Pages:
119–129 (11 total)
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Nature of ligand affinity and dimerization of corticotrophin-releasing factor-binding protein may be detected by circular dichroism

P J Lowry, S C Koerber, R J Woods, S Baigent, S Sutton, D P Behan, W Vale, and J Rivier

ABSTRACT

As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the α-helical CRF(9–41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly β-sheet and the peptides showing mainly α-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in α-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.

DOI:
https://doi.org/10.1677/jme.0.0160039
Volume 16: Issue 1,
Pages:
39–44 (6 total)