Recombinant Melittin Research Articles

Construction, cloning, and expression of Melittin antimicrobial peptide using Pichia pastoris expression system

With the emergence of antibiotic resistance, researchers have recently focused on antimicrobial peptides as potential drug candidates. In this study, Melittin, one of the most effective antimicrobial peptides of honey bee venom was produced by the expression system of Pichia pastoris GS115. Melittin was cloned into pPIC9 vector, transformed, and integrated into the P. pastoris genome. The highest level of expression was observed in BMM minimal medium without protein, pH 6, 2% methanol, and 48 h. Then, the recombinant Melittin was tested on some gram-positive and gram-negative bacteria to investigate the antimicrobial effects and showed wide-spectrum activity. The production of Melittin by this method costs very low and paves the way for further studies and also therapeutic usages.

Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy.

The expression and purification of melittin (MET) in microbials are difficult because of its antibacterial activities. In this work, MET was fused with a glutathione-S-transferase (GST) tag and expressed in Escherichia coli to overcome its lethality to host cells. The fusion protein GST-MET was highly expressed and then purified by glutathione sepharose high-performance affinity chromatography, digested with prescission protease, and further purified by Superdex Peptide 10/300 GL chromatography. Finally, 3.5 mg/L recombinant melittin (rMET) with a purity of >90% was obtained; its antibacterial activities against Gram-positive Bacillus pumilus and Staphylococcus pasteuri were similar to those of commercial MET. A circular dichroism spectroscopic assay showed that the rMET peptide secondary structure was similar to those of the commercial form. To our knowledge, this is the report of the preparation of active pure rMET with no tags. The successful expression and purification of rMET will enable large-scale, industrial biosynthesis of MET.

High-Level Expression and Purification of Melittin in Escherichia coli Using SUMO Fusion Partner

Melittin (MLT) is a small cationic peptide discovered from the bee venom. It is used as an antimicrobial agent due to its broad-spectrum activities against bacteria, fungi and tumor cells. But the sources limit its applications. Therefore, the small ubiquitin-related modifier (SUMO) fusion technology was reported for high-level expression of Melittin. pET-3c-SUMO-Melittin plasmid was constructed, and the fusion protein (SUMO-Melittin) was expressed in a soluble form and purity by Ni2+-NTA chromatography. After the SUMO-Melittin fusion protein was cleaved by the SUMO protease, the cleaved sample was purified again by a Ni2+-NTA. Finally, about 25 mg recombinant Melittin was obtained from 1L fermentation culture with more than 95% purity. The recombinant Melittin exhibited similar cytotoxicity and antimicrobial properties as the synthetic Melittin. Thus, a system for high-level expression of Melittin was successfully established in this study and could be used for preparation of similar antimicrobial peptides.

Helical Structure of Recombinant Melittin.

Melittin is an extensively studied, 26-residue toxic peptide from honey bee venom. Because of its versatility in adopting a variety of secondary (helix or coil) and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies as well as in studies involving binding to proteins, lipids, and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of trifluoroethanol (TFE): a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using nuclear Overhauser effect (NOE)-derived distance restraints from three-dimensional NMR spectra, we determined the atomic resolution solution NMR structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFE-containing medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N- and C-terminal α-helices and the noncanonical 310-helix in the middle. The noncanonical 310-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin's structure in TFE-containing medium provides insights into melittin's conformational transitions, which are relevant to the peptide's interactions with its biological targets.

Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

In recent years, there has been a resurgence of interest in melittin and its variants as their therapeutic potential has become increasingly evident. Melittin is a 26-residue peptide and a toxic component of honey bee venom. The versatility of melittin in interacting with various biological substrates, such as membranes, glycosaminoglycans, and a variety of proteins, has inspired a slew of studies that aim to improve our understanding of the structural basis of such interactions. However, these studies have largely focused on melittin solutions at high concentrations (>1 mM), even though melittin is generally effective at lower (micromolar) concentrations. Here we present high-resolution nuclear magnetic resonance studies in the lower-concentration regime using a novel method to produce isotope-labeled (15N and 13C) recombinant melittin. We provide residue-specific structural characterization of melittin in dilute aqueous solution and in 2,2,2-trifluoroethanol/water mixtures, which mimic melittin structure-function and interactions in aqueous and membrane-like environments, respectively. We find that the cis-trans isomerization of Pro14 is key to changes in the secondary structure of melittin. Thus, this study provides residue-specific structural information about melittin in the free state and in a model of the substrate-bound state. These results, taken together with published work from other laboratories, reveal the peptide's structural versatility that resembles that of intrinsically disordered proteins and peptides.

Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation.

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ∼ 0.5-1 mg/L of E. coli culture.

Proteolytically activated anti-bacterial hydrogel microspheres

Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released.

Anti-proliferative activity of recombinant melittin expressed in Escherichia coli toward U937 cells

Melittin, a toxic peptide found in bee venom, has been widely reported to have antimicrobial and anti-proliferative activity in many cell lines. While high-purity natural melittin is rare and expensive, the demand for melittin has increased, making it urgent to find a cheap, suitable method of mass production. In this study, the melittin gene of the Chinese bee (honeybee) was synthesized according to the sequence published in GenBank and cloned into the prokaryotic expression vector pGEX-6P-1. Recombinant melittin was then successfully expressed in Escherichia coli. The activity of affinity-purified recombinant melittin was determined in human leukemic U937 cells. Results show that the recombinant melittin had the same anti-proliferative activity in human leukemic U937 cells in vitro as natural one. This shows the promise of recombinant melittin as a replacement for the natural bee toxins used in drug development and other applications. Key words : Escherichia coli, melittin, expression, anti-proliferative activity.

Expression of the melittin gene of Apis cerana cerana (Hymenoptera: Apidae) in insect cells

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.

Expression of the melittin gene of Apis cerana cerana in Escherichia coli

A cDNA encoding melittin in Apis cerana cerana was obtained by PCR from the recombinant plasmid and cloned into the GST fusion expression vector pGEX-4T-2 for expression of the protein. The expressed protein of about 29 kDa was detected by Western blot and triple antibody sandwich ELISA, indicating that the recombinant protein is the fusion protein of GST-AccM. The expression conditions of GST-AccM fusion protein for Escherichia coli BL21 transformant were optimized. Thin layer scanning on the SDS–PAGE profiles of GST-AccM showed that the expressed protein accumulated up to about 15.2% of total protein of bacterial cells under the optimized expression condition. Purified and recovered recombinant melittin of A. cerana cerana showed bioactivity in activating rabbit platelets to aggregate.