Abstract
A fragment encoding melittin cDNA from Apis cerana cerana fused with glutathione S-transferase gene was inserted into the multiple cloning site of the pBacFastHTb to construct a recombinant donor plasmid, pBacHT-GSTAccM, which was transposed to the target bacmid in E. coli (DH10) by Tn7 transposition function. Then the recombinant baculovirus Bacmid-GSTAccM was transfected into Tn-5B1-4 cells of the cabbage looper, Trichoplusia ni, mediated by lipofectin. The expressed protein of about 34 kDa was detected by Western blotting and triple antibody sandwich ELISA, indicating that the recombinant protein is the fusion protein of GSTAccM. Thin layer scanning showed that the expression level of GSTAccM was about 7% of the total cell protein. Purified and recovered recombinant melittin of A. c. cerana showed bioactivity in activating rabbit platelets to aggregate.
Highlights
The honeybee, Apis mellifera L., venom gland produces an aqueous secretion, which contains a significant quantity of two enzymes and four peptides
We report the successful expression of the melittin gene from the Chinese honeybee as a glutathione S-transferase (GST) fusion protein in baculovirus-infected insect cells
The melittin gene of the Chinese honeybee was successfully expressed in insect cells using the Bac-to-Bac® baculovirus expression system
Summary
The honeybee, Apis mellifera L., venom gland produces an aqueous secretion, which contains a significant quantity of two enzymes and four peptides. Melittin, comprising 50% of its dry weight, is a basic, amphipathic polypeptide (Pott et al, 1998). It is characterized by its powerful hemolytic activity and a popular model peptide for probing lipid-peptide interactions (Subbalakshmi et al, 1999; Benachir et al, 1997). We explore eukaryotic expression systems based on insect cells for the production of melittin of the Chinese honeybee as these systems have the potential for producing large amounts of correctly processed and secreted proteins. We report the successful expression of the melittin gene from the Chinese honeybee as a glutathione S-transferase (GST) fusion protein in baculovirus-infected insect cells
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