Cloning, Expression And Antimicrobial Activities Of Musca Domestica Cecropin Fused With Human Lysozyme

Cecropin, first isolated from insects, is one of the best characterized AMPs. Cecropin display antimicrobial activities, antiviral activities, mediating cytotoxicity towards tumour cells, immunomodulatory activities and without affecting human normal cells. It is believed that one of the main targets for these peptides is the cytoplasmic membrane. Permeabilization of the membrane by pore formation, allowing the cell contents to be released, finally resulting in cell death. Human lysozyme is a natural antimicrobial protein that occurs in a variety of normal human tissues and secretions. It targets theβ(1–4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making it highly active against Gram positive bacteria. It is found that cecropin act in synergy with lysozyme.This can be explained in part by cecopin disrupt the outer membrane, giving the enzyme access to the peptidoglycan of cell wall. The increasing resistance of bacterial strains to classical antibiotics is a major concern worldwide, leading the antimicrobial protein such as cecropin, lysozyme may have important potential application in the future due to their antimicrobial activities and especial mechanisms of action. In the present study, a novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was constructed by splice-overlap extension-PCR. The DNA sequence encoding recombination fusion protein Mdc-hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). Their antimicrobial activities were assayed and compared with its parental peptides. The machanisms of action of fusion protein Mdc-hly were examined by scan electron microscopic and transmission electron microscopic. Mdc-hly may lay a solid foundation for the further research to develop a new polypeptide drug with dual action mechanisms.Methods:1. Cloning of Musca domestica cecropin (Mdc) and Human lysozyme (Hly): Total RNA was extracted from the third instar larvae of Musca domestica and human placental with Trizol (Invitrogen) as described by the manufacturer, individual. The Mdc and Hly genes were amplified by reverse-transcription polymerase chain reaction (RT-PCR) based on the nucleotide sequence of Musca domestica cecropin (GenBank Accession No. EF175878) and human lysozyme (GenBank Accession No. NM000239) using specific primers, respectively. The resultant cDNA fragments were cloned into pMD20-T vector to yield recombinant plasmids Mdc/pMD20-T and Hly/pMD20-T. The resulting plasmids were transformed into E. coli DH5a cells to verify by PCR screening, restriction endonuclease analysis and DNA sequencing.2. Recombination of Mdc and Hly gene by SOE-PCR and bioinformatics analysis of fusion Mdc-hly: The fusion gene Mdc-hly was constructed in a Mdc-linker-hly format with standard 15-amino acid linker (Gly4Ser)3 by splice-overlap extension-PCR (SOE-PCR). The final full-length product was cloned into the pMD20-T vector to yield recombinant plasmid Mdc-hly/pMD20-T. To definitively ensure that the gene had been amplified without introducing nucleotide errors, the resulting plasmids were transformed into E. coli DH5a cells to verify by PCR screening, restriction endonuclease analysis and DNA sequencing. The physical-chemical properties, the conserved domains, hydrophobicity, second structure and tertiary structure of fusion protein Mdc-hly were analysised by bioinformatics analysis software.3. Recombinant expression of fusion gene Mdc-hly and purification of fusion protein Mdc-hly: The generated recombinant plasmids Mdc-hly/pMD20-T were digested with NocI and HindIII restriction enzymes and the excised products were subcloned into expression vector pET-32a, previously digested with the same restriction enzymes. The resultant recombinant expression vectors Mdc-hly/pET-32a were finally transformed into E. coli BL21 (DE3) for fusion protein expression. After induction by IPTG, the recombinant Trx-6His-Mdc-hly was analyzed by SDS-PAGE and determined by western blot. The fusion Trx-6His-Mdc-hly was purified by affinity chromatograph column. The Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin.4. Assay of antimicrobial activity and action machanism of fusion protein Mdc-hly: Microdilution assays to establish minimal inhibition concentration (MIC) values and Minimal bactericidal concentration (MBC) of recombination proteins Mdc-hly, Hly and Mdc against E. coli ATCC25922, P. aeruginosa ATCC27853, S. paratyphi-B CICC 21495, S.aureus ATCC25923, B.subtilis ATCC6633, M. lysodeikticus CICC 23645, were performed. Scan electron microscopic and transmission electron microscopic were used to observe the morphological and structure changes of S.aureus ATCC25923 and E. coli ATCC25922 treated with recombination proteins Mdc-hly, Hly and Mdc.Results:1. Cloning of Musca domestica cecropin (Mdc) and Human lysozyme (Hly): Total RNA of the third instar larvae of Musca domestica and human placental were used as templates for cDNA synthesis. The DNA fragment encoding the mature protein of Musca domestica cecropin and human lysozyme were obtained by RT-PCR and the resultant cDNA fragment was cloned into pMD20-T vector. Mdc with about 140 bp and Hly with about 410 bp were verified by PCR screening, restriction endonuclease analysis and sequencing. The results confirmed that the senquences of Mdc and Hly were identifying to the sequences reported in Genbank.2. Recombination of Mdc and Hly gene by SOE-PCR and bioinformatics analysis of fusion Mdc-hly: With SOE-PCR method, the full-length Mdc-hly gene wtih a hydrophobic polypeptide linker (Gly4Ser)3 was obtained and the sequence had been verified without introducing nucleotide errors by PCR screening, restriction endonuclease analysis and sequencing. Bioinformatics analysis results indicate that Mdc-hly is cationic molecules with molecular weight of 20131.7 Da, theoretical PI of 9.69, containing the conserved domains of both Mdc and Hly. Instability index classifies the protein as stable, and the second structures of Mdc-hly containα-helix,β-turn and random coil.3. Recombinant expression of fusion gene Mdc-hly and purification of fusion protein Mdc-hly: The Mdc-hly gene fragment was cloned into expression vector pET-32a. The prokaryotic expression plasmids Mdc-hly/pET-32a were constructed successfully, which confirmed by PCR screening, restriction endonuclease analysis and sequencing. The resultant recombinant expression plasmids Mdc-hly/pET-32a were transformed into E. coli BL21 (DE3). After induction by IPTG, the recombinant Trx-6His-Mdc-hly was highly and inducibly expressed in an insoluble form. SDS-PAGE and western blot analysis confirmed that the 38 kDa protein was the fusion Trx-6His-Mdc-hly. Using HisTrap HP affinity column, the fusion Trx-6His-Mdc-hly was easily purified. The Mdc-hly was released from the fusion Trx-6His-Mdc-hly by enterokinase cleavage and separated from the carrier thioredoxin. Approximately 21.4 mg of the pure recombinant Mdc-hly was obtained from 1 L of E. coli cultrue4. Assays of antimicrobial activity and action machanism of fusion protein Mdc-hly: Antimicrobial activity assays showed that Mdc has activity against both Gram-negative and Gram-positive bacteria, and the Gram-negative strains were more sensitive to Mdc than the Gram-positive strains. Hly showed activity against Gram-positive bacterium, whereas Hly did not inhibit the growth of the tested Gram-negative strains under the same conditions. The recombinant fusion protein Mdc-hly was improved in vitro antimicrobial activity and action spectrum compared to Mdc and Hly. The results of scaning electron microscope showed that: The bacteria outer membrane was achieved with a loss in continuity when bacteria cells were incubated with Mdc; A remarkable damage in the bacteria cell walls when bacteria cells were incubated with Hly; Cells showed disintegration of the cells and extensive damage of the cell membranes and cell walls when bacteria cells were incubated with fusion protein Mdc-hly. The results of transmission electron microscope showed that: Bacterial cells treated with Mdc showed loss of outer membrane, cytoplasm condensation and karyopyknosis; Bacterial cells treated with Hly showed cell walls missing; A great number of cells appeared with their cell walls missing, extensive damaging of the cell membranes, the cytoplasmic content leaked to the extracellular medium and disintegration of the cells when bacteria cells were incubated with fusion protein Mdc-hly. These results indicated that the main target for Mdc is the cytoplasmic membrane as typical insect cecropins; the main target for Hly is cell wall; while the fusion protein Mdc-hly exerts its antimicrobial activity by acting on both the plasma membrane and the cell wall.Conclusions:The DNA fragment encoding the mature protein of Musca domestica cecropin and human lysozyme were obtained by RT-PCR, which identify to the sequences reported in Genbank website. The Mdc-hly gene was successfully constructed in a Mdc-linker-hly format with the standard 15-amino acid linker (Gly4Ser)3 by SOE-PCR method. Bioinformatics analysis results indicate that the fusion protein Mdc-hly containing the conserved domains of both Mdc and Hly, and with the physical-chemical properties of the antimicrobial proteins. The prokaryotic expression plasmids Mdc-hly/pET-32a were constructed successfully and transformed into E. coli BL21 (DE3) for protein expression. After induction by IPTG, the recombinant Trx-6His-Mdc-hly was highly expressed. The fusion Trx-6His-Mdc-hly was purified by affinity chromatography, and the Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. The recombinant fusion protein Mdc-hly was improved in vitro antimicrobial activity and action spectrum compared to Mdc and Hly. To further inverstigate the mechanisms of action of fusion protein Mdc-hly, ultraxtructural observation of S. aureu and E. coli treated with Mdc-hly, Mdc, Hly on Scanning Electron Microscope and Transmission Electron Microscope were performed. The results showed that the fusion protein Mdc-hly exerts its antimicrobial activity by acting on both the plasma membrane and the cell wall. These studies provide a simple and reliable method to produce a large quantity of biologically active antimicrobial proteins in E.coli cell, which can be used for structural and functional analysis and clinical applications. The fusion protein may have important potential application as a future safely administered human drug and food additive.