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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., (© 2023. The Author(s).)

Due to the increasing global demand for Gamma-aminobutyric acid (GABA) in the food and pharmaceutical industries, the expression of the recombinant Glutamate decarboxylase (GAD) and its industrial production is currently requested. Culture conditions were optimized to increase the expression level of the recombinant enzyme in different pH, temperature, incubation time, aeration levels, inoculation concentrations, concentrations of IPTG, and several carbon sources using the RSM based on a central composite design. According to the results of the quadratic regression equation, recombinant Escherichia coli BL21 (DE3) had the highest GAD expression at pH=7.2, aeration at about 120 rpm, inoculation concentrations of about 3% v/v, 2.25 mM IPTG, 37 °C, and 6 h of incubation time in the presence of 0.2% glucose. Using pure glucose as a carbon source on an industrial scale is not cost-effective for producing recombinant proteins. Therefore, using low-cost carbon sources such as corn syrup and molasses with concentrations of 1.5 and 5.65% (w/v) is an efficient method for the industrial production of recombinant GAD. The concentration of purified recombinant GAD in carbon sources of 0.2% glucose, 1.5 corn syrup and 5.65% molasses was 2.155, 2.07 and 1.96 mg/mL, respectively. In this way, the global need for GABA can be met by the industrial production of GAD. [ABSTRACT FROM AUTHOR]

The effect of different levels of Scrophularia striata extract in normal and challenged conditions with E. coli on performance, carcass characteristics, cellular immune response, blood antioxidant status, intestinal histo-morphometry, and microbial population (E.coli and Lactobacillus) of Cobb 500 broilers were evaluated. The experiment was performed in a complete block design with a 2 × 4 factorial arrangement with five replications of 10 birds each for 35 days in two separated halls [include challenge (C) and non-challenge (N)] with similar experimental conditions. Experimental treatments in two breeding halls were: 1) basal diet or control (CONT), 2) basal diet + 0.1 g/kg of herbal extract (EXTR 1), 3) basal diet +0.2 g/kg of herbal extract (EXTR 2), 4) Basal diet +0.1 g/kg an antibiotic (Oxytetracycline) (ANTB). On the 16th and 24th days of the experiment, one dose of 1×107 CFU K99 E. Coli was gavaged to chickens in a challenged conditioned hall. The results showed that average body weight and daily weight gain in the whole period (days 35 and 1-35) in EXTR 2 was better than the control treatment (P < 0.05). The percentage of breast weight in the ANTB was significantly different from the control treatment (P < 0.05). Glutathione peroxidase (GPX) in EXTR 2 was better than the control treatment (P < 0.05). Malondialdehyde (MDA) in EXTR 1 was significantly lower than the control group (P < 0.05). The values of E. coli in the treatment ANTB were less than control treatment (P < 0.05). Lactobacillus value in treatment EXTR 2 was higher than control treatment (P < 0.05). The value of total immunoglobulin in 28 days in ANTB, EXTR 1, and EXTR 2 were significantly higher than that of control treatment (P < 0.05). The length of villi was affected by treatments (P < 0.05). In conclusion, dietary inclusion of 0.2 g/kg of Scrophularia striata extract may improve the health status of the birds during E. coli challenge. [ABSTRACT FROM AUTHOR]

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD–thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 μg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 μg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD–thanatin interaction. [ABSTRACT FROM AUTHOR]

Computational prediction of signal peptides is one of the most important steps in genetic engineering experiments. The periplasmic expression cause the reducing in the inherent destructive behavior of Bofurin I against its host and also reducing its susceptibility to proteolytic degradation. In order to predict the best signal peptides for expression of Buforin I in E. coli, 103 signal sequences were retired from signal peptide databases. Since the purpose of this study was to introduce the optimal signal peptides for periplasmic expression, first, sub-cellular localization site of signal peptides was analyzed. Then, n, h, and c regions of signal peptide, signal peptide probability and physico-chemical features were investigated. Base on the results, MalE, hofQ, papK, ugpB, zraP, and sfmC were introduced as the best signal peptides. For increasing the half-life of mRNA and the increasing the stability of the mRNA against exonuclease activity, secondary structures of mRNA including Shine-Dalgarno, untranslated region of ompA, start codon, signal peptide and sequences of Buforin I were analyzed. Based on the total free energy pilot evaluated and mRNA conformations, papK seemed more appropriate than the rest of the signal peptides. The obtained result of this study can be used for design the periplasmic expression constructs. [ABSTRACT FROM AUTHOR]

Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the decarboxylation reaction, which is catalyzed by the glutamate decarboxylase (GAD). Escherichia coli is widely being used to express proteins. However, without appropriate signal peptide, it cannot be applied for secretory proteins. Selecting a suitable signal peptide (SP) is a critical step in the secretory production of different proteins. In silico identification of suitable SP is a reliable and cost-effective alternative to experimental approaches. In previous studies, the localization of proteins was not considered and the SPs of periplasmic, membranes and extracellular were compared. Therefore, this study aimed to predict the best SP for the expression of recombinant GAD in the outer membrane of E. coli only. Also, we compared twelve servers to evaluate protein localization, solubility, and secretory pathway. In the present study, 127 SPs were taken from the Signal Peptide database. The localization site, physico-chemical properties, location of cleavage sites, regions and D-score of them were determined by ProtComp, ProtParam, and SignalP 3.0 and 4.1 servers, respectively. To rank SPs based on the secretion properties, PRED-TAT and SignalP 5.0 webservers were used. Based on the results, the localization site of 13 SPs was in the outer membrane of E. coli. Among them, the most suitable candidates seemed to be torT with a reasonably high D-score, aliphatic index, and GRAVY, followed by ccmH and then pspE. TorT accelerates GAD scale-up production and might be useful in future experimental research. [ABSTRACT FROM AUTHOR]

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics. [ABSTRACT FROM AUTHOR]