Molecular cloning and characterization of a recombinant Bombyx mori tyramine-β-hydroxylase in a silkworm cell line using a baculovirus expression vector system

Ahmed M.H. Ali, Nazmul Hasan, Renkai Guo, Hiroto Ohta, Akinori Hirashima

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesis of OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkworm B. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frame encoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belonging to the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expression vector system. Western blot analysis revealed an immunoreactive band with a molecular mass of ~. 67.4. kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the products formed during incubation of the enzyme reaction mixture. The optimum pH and temperature for the conversion of TA to OA were 7.5 and 25. °C, respectively. During incubation, the reaction was linear for the first 30. min at 25. °C and pH. 7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed that this method can be used for screening of BmTβH inhibitors.

Original languageEnglish
Pages (from-to)221-227
Number of pages7
JournalJournal of Asia-Pacific Entomology
Volume17
Issue number3
DOIs
Publication statusPublished - Sep 1 2014

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tyramine
Baculoviridae
Bombyx mori
silkworms
molecular cloning
cell lines
octopamine
neurotransmitters
neurohormones
reversed-phase liquid chromatography
biogenic amines
enzymatic reactions
sequence homology
nervous system
open reading frames
Western blotting
amino acid sequences
high performance liquid chromatography
nucleotides
invertebrates

All Science Journal Classification (ASJC) codes

  • Insect Science

Cite this

Molecular cloning and characterization of a recombinant Bombyx mori tyramine-β-hydroxylase in a silkworm cell line using a baculovirus expression vector system. / Ali, Ahmed M.H.; Hasan, Nazmul; Guo, Renkai; Ohta, Hiroto; Hirashima, Akinori.

In: Journal of Asia-Pacific Entomology, Vol. 17, No. 3, 01.09.2014, p. 221-227.

Research output: Contribution to journalArticle

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