title: Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae.
abstract: CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/α Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120 h, the maximum amount of total secreted protein was 1.437 g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81 μM. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200 μM. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 °C to 100 °C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy.
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, PR China.
authors.forename: Lijie
authors.initials: L
authors.lastname: Xia
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Liu
authors.forename: Ji
authors.initials: J
authors.lastname: Ma
authors.forename: Surong
authors.initials: S
authors.lastname: Sun
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yang
authors.forename: Fuchun
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology; D012831:Bombyx / Q000378:metabolism*; D054807:Cecropins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000900:Anti-Bacterial Agents; D054807:Cecropins; D011994:Recombinant Proteins