Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli.

The lack of cost-effective methods for producing antimicrobial peptides has made it impossible to use their high potential as a new and powerful class of antimicrobial agents. In recent years, extensive research has been conducted to decrease the cost of recombinant proteins production through microorganisms, transgenic animals, and plants. Well-known genetic and physiological characteristics, short-term proliferation, and ease of manipulation make E. coli expression system a valuable host for recombinant proteins production. Expression in periplasmic space is recommended to reduce the inherently destructive behavior of antimicrobial peptides against the expressing microorganism and to decline susceptibility to proteolytic degradation. In this study, a pET-based expression system was used to express buforin I at E. coli periplasmic space, and its antimicrobial, hemolytic, and cell toxicity activities as well as structural stability were evaluated. The hemolysis activity and cytotoxicity of His-tagged buforin I were negligible and its antimicrobial activity did not show a significant difference compared to synthetic buforin I. In addition, in silico investigating of stability of native and His-tagged buforin I showed that RMSF, RMSD and Rg curves had followed a similar trend during 150 ns simulation. Furthermore, evaluating the modelled structures, FTIR and X-ray methods of both peptides indicated an insignificant structural difference. It was concluded that the recombinant buforin I could be a viable alternative to some currently used antibiotics by successfully expressing it in the pET-based expression system.

Preparation and in vitro evaluation of injectable formulations of levothyroxine sodium using in situ forming hydrogel temperature-responsive systems based on PLA-PEG-PLA and PLGA-PEG-PLGA triblock copolymers.

Objectives: Recently, great attention has been paid to developing new drug delivery systems to manage the rate, time, and site of drug release. We aimed to design a novel drug delivery system to support targeted and gradual delivery of levothyroxine sodium. Materials and Methods: The triblock copolymers of PLA-PEG-PLA and PLGA-PEG-PLGA were constructed using the ring-opening copolymerization method and then purified and characterized by 1H-NMR, DSC, and GPC techniques. The phase transition temperature of the polymers was determined, and levothyroxine sodium stability was investigated in a phosphate-based buffer (pH 7.4). In vitro drug release into the PBS was measured at different concentrations of the triblocks for one month. Results: The results of NMR and GPC showed successful fabrication of the copolymers with low molecular weight dispersion and Tg points of -8.19 °C and -5.19 °C for PLA-PEG-PLA and PLGA-PEG-PLGA, respectively. Stability tests showed that during one month, most of the triblocks' masses degraded at 37 °C while levothyroxine sodium remained stable. Initial burst release of the drug in both copolymers is inversely correlated with the concentration of the polymer. Evaluation of drug release for 35 days showed that PLA-PEG-PLA had a slower drug release rate than PLGA-PEG-PLGA. Conclusion: Considering the low initial burst release, as well as continuous and long-term release kinetics of PLA-PEG-PLA and PLGA-PEG-PLGA copolymers, they can be used to gradually deliver levothyroxine sodium, obviating the need for frequent administrations and concerns over drug-food interactions.

Buforin I an alternative to conventional antibiotics: Evaluation of the antimicrobial properties, stability, and safety.

Cationic antimicrobial peptides are being developed as a promising class of antimicrobial sub-stances. The introduction of a new antibiotic component requires a comprehensive study of its properties so that it can be relied upon to continue laboratory procedures and clinical trials on laboratory animals or human volunteers. Antimicrobial activity of buforin I was evaluated against 15 of the most important pathogenic bacterial and fungal strains. This was followed by assessing anti-biofilm activity, time-dependent inhibitory, thermal stability, plas-ma stability, hemolysis, and cytotoxic activities. The range of obtained MICs was between 4 and 16 µg/mL. The most resistant and most sensitive microbial strains were S. salivarius and C. perfringens, respectively. Buforin I not only inhibited biofilm formation, but also showed a high biofilm radiation activity. Buforin I was stable in human plasma and also at different temperatures including 40, 60, and 80 °C. Although no significant anti-cancer properties were observed for buforin I, the lack of cytotoxicity as well as the lack of hemolytic activity confirm its safety. The high therapeutic index indicated that buforin I has a considerable pharmaceutical potential and can be a reasonable candidate to replace antibiotics or administered in combination with antibiotics to increase the effectiveness as well as reduce the dose of antibiotics.

Evaluation of Antimicrobial Activity of Buforin I and Nisin and Synergistic Effect of the Combination of them as a Novel Antimicrobial Preservative.

One of the most effective methods for increasing the antimicrobial activity of a substance is to combine it with one or more other antimicrobial agents. The aim of the present study was to evaluate the antimicrobial effect of buforin I and nisin alone and investigate the synergistic action of these compounds against the most important food spoilage microorganisms in clouding B. subtilis, S. epidermidis, L. innocua, E. coli, S. Enteritidis, A. oryzae, R. glutinis and G. candidum. The results of MIC and MBC/MFC examinations showed that buforin I had higher antimicrobial activity than nisin on all the microbial strains used in this study (p≤0.5). E.coli was the most resistant to both antimicrobial agents, while Listeria innocua and Staphylococcus epidermidis were the most sensitive to nisin and buforin I, respectively. The results of synergistic interaction between buforin I and nisin indicated that the combination of buforin I and nisin on B. subtilis, S. epidermidis and A. oryzae showed synergistic effect, while it had no effect on S. Enteritidis and Geotrichum candidum. The combination of buforin I and nisin showed partial synergistic effect on Listeria innocua, Escherichia coli, Rhodotorula glutinis. Assessment of viability of the microorganisms under the antimicrobial agents alone and in combination with each other at MICs and FICs indicated that use of these antimicrobial agents in combination enhances antimicrobial activity at lower concentrations of both agents. The present study investigated the antimicrobial properties of buforin I against food spoilage microorganisms for the first time and suggests that its use alone or in combination with nisin may provide a clear horizon for the application of antimicrobial peptides as natural preservatives. Thus, the combination of antimicrobial peptides and traditional antimicrobial food preservative could be a promising option for the prevention of contamination, spoilage, and infestation of food and beverage products.