Cloning, recombinant expression, purification, and functional characterization of AGAAN antibacterial peptide.

A recombinant version of the AGAAN antimicrobial peptide (rAGAAN) was cloned, expressed, and purified in this study. Its antibacterial potency and stability in harsh environments were thoroughly investigated. A 15 kDa soluble rAGAAN was effectively expressed in E. coli. The purified rAGAAN exhibited a broad antibacterial spectrum and was efficacious against seven Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration (MIC) of rAGAAN against the growth of M. luteus (TISTR 745) was as low as 60 µg/ml. Membrane permeation assay reveals that the integrity of the bacterial envelope is compromised. In addition, rAGAAN was resistant to temperature shock and maintained a high degree of stability throughout a reasonably extensive pH range. The bactericidal activity of rAGAAN ranged from 36.26 to 79.22% in the presence of pepsin and Bacillus proteases. Lower bile salt concentrations had no significant effect on the function of the peptide, whereas higher concentrations induced E. coli resistance. Additionally, rAGAAN exhibited minimal hemolytic activity against red blood cells. This study indicated that rAGAAN may be produced on a large scale in E. coli and that it had an excellent antibacterial activity and sufficient stability. This first work to express biologically active rAGAAN in E. coli yielded 8.01 mg/ml at 16 °C/150 rpm for 18 h in Luria Bertani (LB) medium supplemented with 1% glucose and induced with 0.5 mM IPTG. It also assesses the interfering factors that influence the activity of the peptide, demonstrating its potential for research and therapy of multidrug-resistant bacterial infections.

Recombinant Active Peptides and their Therapeutic Functions.

Recombinant active peptides are utilized as diagnostic and biotherapeutics in various maladies and as bacterial growth inhibitors in the food industry. This consequently stimulated the need for recombinant peptides' production, which resulted in about 19 approved biotech peptides of 1- 100 amino acids commercially available. While most peptides have been produced by chemical synthesis, the production of lengthy and complicated peptides comprising natural amino acids has been problematic with low quantity. Recombinant peptide production has become very vital, costeffective, simple, environmentally friendly with satisfactory yields. Several reviews have focused on discussing expression systems, advantages, disadvantages, and alternatives strategies. Additionally, the information on the antimicrobial activities and other functions of multiple recombinant peptides is challenging to access and is scattered in literature apart from the food and drug administration (FDA) approved ones. From the reports that come to our knowledge, there is no existing review that offers substantial information on recombinant active peptides developed by researchers and their functions. This review provides an overview of some successfully produced recombinant active peptides of ≤100 amino acids by focusing on their antibacterial, antifungal, antiviral, anticancer, antioxidant, antimalarial, and immune-modulatory functions. It also elucidates their modes of expression that could be adopted and applied in future investigations. We expect that the knowledge available in this review would help researchers involved in recombinant active peptide development for therapeutic uses and other applications.

Antibacterial Potential of a Novel Peptide from the Consensus Sequence of Dermaseptin Related Peptides Secreted by Agalychnis annae.

BACKGROUND: The consistently increasing reports of bacterial resistance and the reemergence of bacterial epidemics have inspired the health and scientific community to discover new molecules with antibacterial potential continuously. Frog-skin secretions constitute bioactive compounds essential for finding new biopharmaceuticals. The exact antibacterial characterization of dermaseptin related peptides derived from Agalychnis annae, is limited. The resemblance in their conserved and functionally linked genomes indicates an unprecedented opportunity to obtain novel bioactive compounds. OBJECTIVE: In this study, we derived a novel peptide sequence and determined its antibacterial potentials. METHODS: Consensus sequence strategy was used to design the novel and active antibacterial peptide named 'AGAAN' from skin secretions of Agalychnis annae. The in-vitro activities of the novel peptide against some bacterial strains were investigated. Time kill studies, DNA retardation, cytotoxicity, betagalactosidase, and molecular computational studies were conducted. RESULTS: AGAAN inhibited P. aeruginosa, E. faecalis, and S. typhimurium at 20 µM concentration. E. coli and S. aureus were inhibited at 25 µM, and lastly, B. subtilis at 50 µM. Kinetics of inactivation against exponential and stationary growing bacteria was found to be rapid within 1-5 hours of peptide exposure, depending on time and concentration. The peptide displayed weak hemolytic activity between 0.01%-7.31% at the antibacterial concentrations. AGAAN efficiently induced bacterial membrane damage with subsequent cell lysis. The peptide's DNA binding shows that it also targets intracellular DNA by retarding its movement. Our in-silico molecular docking analysis displayed a strong affinity to the bacterial cytoplasmic membrane. CONCLUSION: AGAAN exhibits potential antibacterial properties that could be used to combat bacterial resistance.