Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori

Abstract

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

Bioactive Constituents of the Silk Worm Bombyx mori

“…Two foreign gene expression systems in Escherichia coli were adopted to procure a sizeable amount of moricin to facilitate further studies [15]. In addition, Li et al isolated a 35-residue cationic, amphipathic α-helical peptide, named CM4 (ABP-CM4) from silkworm, and this peptide exhibited broad-spectrum antimicrobial activity [16]. Interestinly, recombinant CM4 also showed antimicrobial activity against E. coli, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii [16].…”

Efficient Expression of Bioactive Human Leptin in Escherichia coli in Soluble Fusion Form

“…In fact, it was reported that the solubility of foreign proteins was increased by using of Trx as a gene fusion partner. The use of a Trx fusion partner has been successfully applied to expression of many foreign proteins in soluble form including antibacterial peptide ABP-CM4 [15], FNII-2 [16], and arginyl-tRNA synthetase [17]. So, here, we put the Trx structure at the N-terminus of the rhLeptin; and then we expressed rhLeptin as Trx-fusion forms in E. coli.…”

Antihyperglycemic Effect of Silkworm Powder, Fibroin and Sericin from Three Thai Silkworm (Bombyx mori Linn.) in Streptozotocin-Induced Diabetic Rats

“…2 Mulberry silkworm (B. mori) has been reared for longtime in many regions of Thailand especially the Northeast. 3 Reports on the various pharmacological activities of the silkworm have been accumulated such as antioxidant, 4 anticancer, 5 antibacterial, 6 antiviral, 7 antihypertensive, 8 and hepatoprotective activities. 9 Type 2 diabetes is a chronic disease with the mechanisms potentially linked to overweight and obesity.…”