Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

Enhanced antioxidant properties of sericin-cecropin fusion protein against oxidative stress in human adult dermal fibroblasts

Chronic exposure to UVB radiation causes photoaging, immunosuppression, and ultimately photocarcinogenisis through the generation of reactive oxygen species (ROS). The ability of natural compounds in neutralizing the effects of oxidative stress is being explored with increased interest. Silk sericin, a biopolymer is reported to have diverse biological properties. In an effort to make the silk sericin pure, more effective and multifunctional, we have recombinantly expressed both functional sericin as well as sericin-cecropin B fusion proteins. Herein, we studied the antioxidant and anti-UVB potential of recombinant sericin and sericin-cecropin B proteins against oxidative stress using human primary dermal fibroblast cells. Treating the cells with recombinant sericin (RS) or sericin-cecropin B (RSC) prior to exposure to UVB and H2O2, effectively increased the cell viability by approximately 30% and 50%, respectively, in comparison to non-treated control. The protective effects were further evident in terms of significant reduction of LDH in oxidatively challenged cells treated with RS and RSC. A reduction in LDH release of at least 16 and 33% was observed with RS and RSC treatments, respectively, in comparison to exposed control. Further, elevated levels of catalase and superoxide dismutase (SOD) activity were observed. Importantly, the RSC fusion protein exhibited enhanced protective effects than cells treated with RS alone. Our results demonstrate that the functional attributes of cecropin B along with sericin activity in the fusion protein conferred enhanced protection against UVB- and H2O2-induced oxidative damage in human dermal fibroblasts. The improved antioxidant activity of recombinant sericin fusion biopolymer has great potential as a promising therapeutic agent for ROS-induced skin diseases.

Study of anticancer activity of cecropin B on 7, 12-dimethylbenz (a) anthracene-induced breast cancer

Background and aims: Antimicrobial peptides constitute a family of bioactive peptides that are involved in the body defense. Recently, their anti-cancer properties, especially by inducing apoptosis, have been proven in in vitro studies. Therefore, in this study, the effects of cecropin B as an antimicrobial peptide on breast cancer growth, hematological parameters, and histopathological changes in rats were evaluated. Methods: Twenty-four female rats were randomly divided into 4 groups. The cancer group, control group, cecropin B group, and cancer group treated with cecropin B. The tumor size was measured at the beginning and the completion of the treatment period. Blood samples were collected for assessment of the hematological parameters and Bax and Bcl2 levels. Tumor tissues were removed for histopathological analysis. Results: The tumor size had a significant increase in the cancer group and cancer group treated with cecropin at the end of the treatment. A significant decrease in mean cell volume, white blood cell count and Bcl2 level and a significant increase in hemoglobin and Bax levels were observed in the cancer group treated with cecropin B compared to cancer group. Changes in other parameters were not significant. Histopathological study showed the invasion of mitotic cells to stromal and muscular tissues of the breast in the cancer group, while focal destruction of tissue and cell death were observed in the cancer group treated with cecropin B. Conclusion: The results showed that cecropin B has been able to reduce tumor growth and have little side effects on hematologic factors probably through apoptosis.