Molecular design and biological activity analysis of antimicrobial peptide RIKL / 生物工程学报

Natural antimicrobial peptides have strong bactericidal activities. An obstacle of the development of antimicrobial peptides resides in the difficulty of developing peptides with high biocompatibility. In this study, molecular dynamics analysis was employed to assess the structural characteristics and biological activities of peptides. A (RXKY)2(YRY)2 structure was used as a template to design an antimicrobial peptide RIKL of high-efficiency and low-toxicity, where X represents Ile and Y represents Leu. The secondary structure of the antimicrobial peptide was detected by circular dichroism (CD), and the structures of RIKL in water and in POPC/POPG membrane environment were measured using molecular dynamics. The biological activity of RIKL was further studied by assessing its antimicrobial activity, hemolytic activity, eukaryotic cytotoxicity, and salt ion stability. CD results showed that RIKL presented an α-helical structure in a simulated bacterial membrane environment. Molecular dynamics simulation predicted that the secondary structure of RIKL could be partly retained in water and POPG environment, while this secondary structure was weakened in the POPC environment. Antimicrobial test suggested that RIKL had high antimicrobial activities, and the geometric mean of the Minimum Inhibitory Concentration (MIC) was 3.1 μmol/L. The hemolysis indicated that RIKL had no hemolytic activity within the detection range, and cytotoxicity test suggested the cytotoxicity of RIKL was low. Stability test showed that RIKL maintained antimicrobial activities under different pH, serum concentrations and salt environments. Based on the above results, RIKL has high cell selectivity and has the potential as a highly effective antibacterial drug.

Characteristics of faecal bacterial flora and volatile fatty acids in Min pig, Landrace pig, and Yorkshire pig

BACKGROUND In order to study the influence of long-term growth process and evolution environment on intestinal bacteria of different breeds, the intestinal bacteria and volatile fatty acids among the faeces of Min, Landrace and Yorkshire pigs were analysed by Illumina high-throughput sequencing of the 16S-rDNA and gas chromatography. RESULTS The shared core microbiota of Landrace, Yorkshire and Min pig were 1273, accounting for 69.56% of total abundance of organisms. The proportion of Firmicutes in Min pig faeces (57.89%) was significantly higher than that in Landrace and Yorkshire pig faeces (47.01% and 46.40%, respectively) (P < 0.05), but that of Bacteroidetes was exactly opposite. Moreover, Min pig presented more highly efficient membrane transport, environmental adaptation, carbohydrate transport, and metabolism than Yorkshire pig (P < 0.05). The acetic acid/total volatile fatty acid ratio in Min pig was significantly higher than that in Landrace pig (P < 0.05), and the isobutyric acid/ total volatile fatty acid ratio in Min pig was significantly larger than that in Yorkshire pig (P < 0.05). Furthermore, the content of branched chain volatile fatty acids in Min pig was significantly higher than that in Yorkshire pig (P < 0.05). CONCLUSION This study demonstrated that Min pig, as an excellent breed in the cold area of China, possessed special intestinal floral structure compared to the imported pigs in order to satisfy their phys iological and metabolic demands, which may influence their characteristics such as resistance to cold, diseases, and crude feeding, and the ability to deposit intramuscular fat.

Application of supramolecular peptide self-assembly in biomedicine / 生物工程学报

In recent years, peptide self-assembly has received much attention because of its ability to form regular and ordered structures with diverse functions. Self-assembled peptides can form aggregates with defined structures under specific conditions. They show different characteristics and advantages (e.g., good biocompatibility and high stability) compared with monomeric peptides, which form the basis for potential application in the fields of drug delivery, tissue engineering, and antiseptics. In this paper, the molecular mechanisms, types and influencing factors of forming self-assembled peptides were reviewed, followed by introducing the latest advances on fibrous peptide hydrogels and self-assembled antimicrobial peptides. Furthermore, the challenges and perspectives for peptide self-assembly technology were discussed.

The effects of hinge structure on the biological activity of antimicrobial peptides and its application in molecular design: a review / 生物工程学报

The hinge structure, also known as hinge region or bend, is a special structure found in some antimicrobial peptides. Most studies on antimicrobial peptides focused on the standard secondary structure of α-helix and β-sheet, while the hinge structure and its functions were rarely studied. The hinge structure confers the antimicrobial peptides an improved structural flexibility, which may promote their disruptive effect on bacterial membrane or their binding efficiency to the intracellular targets, thus resulting in a higher antibacterial activity. Meanwhile, the hinge structure may reduce the structural rigidity, which may eliminate the cytotoxicity of antimicrobial peptides to eukaryotic cells. This article reviews the structural characteristics of the hinge structure, its effects on the biological activity of antimicrobial peptides and application in the molecular design, with the aim to provide a reference for the design and development of new antimicrobial peptides.

Design and biological activity of beta-hairpin-like antimicrobial peptide / 生物工程学报

In the current study, we synthesized a 16-residue-long peptide VR with the aim of inspecting the feasibility to design beta-hairpin-like antimicrobial peptide. The peptide was designed by alternating arrangement of arginine and valine and linking two stranded antiparallel beta-sheet with a short loop segment (DPG) and a disulfide bridge. Antimicrobial and hemolytic activities were investigated. Melittin was chosen as a control peptide. We also tested bactericidal kinetics and salt sensitivity. Results show that VR had similar antibacterial activity compared with melittin. However, VR displayed much less hemolytic activity than melittin. These results suggest that VR had higher cell selectivity than melittin. The antibacterial activity of VR was not inhibited in the presence of 25 and 50 mmol/L NaCl. VR still possessed antibacterial activity in the presence of 100 mmol/L NaCl. Collectively, the de novo peptide VR displayed high antimicrobial activity, low hemolytic activity, and salt resistant, indicating that VR was a promising candidate for novel antimicrobial applications.

Application of multi-copies in expression of smaller peptides: a review / 生物工程学报

The technology of genetic engineering has been widely used to express macromolecules such as enzymes. However, it is difficult to detect and purify the micromolecules such as small peptides, because of their instability and degradability. Construction of multi-copy recombinant expression plasmid can be achieved by inserting multiple target genes or expression cassette containing target genes with the same orientation into expression vector. This is effective to increase the expression level of small peptides. In this article we described four methods in order to provide some optional methods and ideas for the expression of active small peptides.

Cloning and expression of a gene encoding shortened LfcinB(1-15)-Melittin(5-12) hybrid peptide in Escherichia coli BL21(DE3) / 生物工程学报

In order to get new antibacterial peptide, we designed a hybrid peptide LfcinB(1-15)-Melittin(5-12), composed of 1-15 amino acid residues of bovine Lactoferricin and 5-12 amino acid residues of Melittin. According to the bias of codon utilization of Escherichia coli, We synthesized the gene encoding the hybrid peptide. We inserted the gene between the sites of Nco I and Sal I of pET-32a and obtained the recombinant expression vector for heterologous expression of LfcinB(1-15)-Melittin(5-12) in Escherichia coli. We used Escherichia coli BL21(DE3) as expression host for the recombinant plasmid. After induced by isopropyl-beta-D-thiogalactoside (IPTG) under the optimized conditions, we realized the fusion protein was successfully expressed. The fusion protein was expressed in soluble form and the level was more than 35% of the total proteins. With (His)6 x Tag, the fusion protein was easily purified by His x Bind Purification Kit. After purification, we obtained 35 mg of fusion protein from 1 L of culture medium. At last, we accomplished that the peptide LfcinB(1-15)-Melittin(5-12) was released from the fusion protein cleaved by enterokinase. The recombinant LfcinB(1-15)-Melittin(5-12) showed antimicrobial activity assayed by agar diffusion test. This is the first report on the heterologous expression of the hybrid antibacterial peptide LfcinB(1-15)-Melittin(5-12) in Escherichia coli and also provides basis for next cost-effective expression of other antimicrobial peptides in genetic engineering.