Cloning, expression, and characterization of diuretic hormone Manduca diuresin from Manduca sexta in Escherichia coli
| Autor: | Asma Ali, Wan Keung Wong, Michael C. Ma |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Transcription
Genetic Recombinant Fusion Proteins Molecular Sequence Data Oligonucleotides Enzyme-Linked Immunosorbent Assay Peptide Biology medicine.disease_cause law.invention law Manduca Escherichia coli medicine Animals Amino Acid Sequence Northern blot Cloning Molecular Gene Chromatography High Pressure Liquid Glutathione Transferase chemistry.chemical_classification Base Sequence Models Genetic Sequence Homology Amino Acid Biological activity DNA Blotting Northern biology.organism_classification Protein Structure Tertiary chemistry Biochemistry Manduca sexta Protein Biosynthesis Recombinant DNA Intercellular Signaling Peptides and Proteins Peptides Biotechnology |
| Zdroj: | Protein Expression and Purification. 29:51-57 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/s1046-5928(03)00007-x |
| Popis: | Manduca diuresin (MD), a 30 amino acid peptide isolated from the tobacco hornworm Manduca sexta, was found to play an important role in the regulation of water and salt balance in the insect. To facilitate studies relating to the function and structure of MD, a synthetic gene encoding MD was assembled and expressed in Escherichia coli. Using an excretion vector, expression of the MD gene in an induced transformant was detected at the transcriptional and translational levels by Northern-blot and ELISA analyses, respectively. With the use of glutathione-S-transferase as the reporter protein, MD was confirmed to be expressed in E. coli. The recombinant product was resolved by reverse-phase HPLC into three peptide groups of different retention times, which were shown by mass spectrometry to be composed of MD deletants missing various lengths of the C-terminus. Despite the deletions and the absence of an amidated C-terminus, the deletants were shown to be biologically active, suggesting the importance of the N-terminus of MD for biological activity. |
| Databáze: | OpenAIRE |
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