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Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs.
- Source :
-
Molecular and cellular endocrinology [Mol Cell Endocrinol] 2006 Jul 11; Vol. 253 (1-2), pp. 96-104. Date of Electronic Publication: 2006 Jun 21. - Publication Year :
- 2006
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Abstract
- A neuropeptide, molt-inhibiting hormone (MIH), negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.
- Subjects :
- Animals
Circular Dichroism
Colforsin analysis
Colforsin chemistry
Colforsin pharmacology
Endocrine Glands chemistry
Endocrine Glands drug effects
Invertebrate Hormones metabolism
Mass Spectrometry
Molting
Nucleotides, Cyclic analysis
Nucleotides, Cyclic chemistry
Nucleotides, Cyclic pharmacology
Peptides chemistry
Protein Folding
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Signal Transduction physiology
Brachyura metabolism
Endocrine Glands metabolism
Escherichia coli genetics
Invertebrate Hormones genetics
Peptides genetics
Peptides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0303-7207
- Volume :
- 253
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Molecular and cellular endocrinology
- Publication Type :
- Academic Journal
- Accession number :
- 16790313
- Full Text :
- https://doi.org/10.1016/j.mce.2006.05.004