Cloning, expression, and characterization of diuretic hormone Manduca diuresin from Manduca sexta in Escherichia coli.
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.
Wong
Wan-Keung R
WK
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. bcwkrw@ust.hk
Ali
Asma B
AB
Ma
Michael C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D018402:Manduca; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D036341:Intercellular Signaling Peptides and Proteins; D009841:Oligonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C072104:Mas-DP II; D004247:DNA; D005982:Glutathione Transferase