Purification Of Antimicrobial Peptides Research Articles

Extraction and Purification of Bioactive Peptide with Antimicrobial Properties from Horseshoe Crab

Abstract: Peptides are small biomolecules that act as bioactive or host defense molecules. These peptides have a significant role in medicine because of their broad spectrum antimicrobial properties. Marine sources are the largest supply of peptides and natural small molecules since they represent half of the total global biodiversity. Among the different crab species, the horseshoe crab is widely used for extracting the peptides. The horseshoe crab is a marine and brackish water arthropod; it plays a crucial role in the reel and whelk fishing industry and the production of important pharmaceutical bioactive components. This review highlights the details of the extraction and purification of antimicrobial peptides from the horseshoe crab. These peptides represent trends and opportunities for the future, with a focus on the pharmaceutical industries.

Bomidin: An Optimized Antimicrobial Peptide With Broad Antiviral Activity Against Enveloped Viruses.

The rapid evolution of highly infectious pathogens is a major threat to global public health. In the front line of defense against bacteria, fungi, and viruses, antimicrobial peptides (AMPs) are naturally produced by all living organisms and offer new possibilities for next-generation antibiotic development. However, the low yields and difficulties in the extraction and purification of AMPs have hindered their industry and scientific research applications. To overcome these barriers, we enabled high expression of bomidin, a commercial recombinant AMP based upon bovine myeloid antimicrobial peptide-27. This novel AMP, which can be expressed in Escherichia coli by adding methionine to the bomidin sequence, can be produced in bulk and is more biologically active than chemically synthesized AMPs. We verified the function of bomidin against a variety of bacteria and enveloped viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), herpes simplex virus (HSV), dengue virus (DENV), and chikungunya virus (CHIKV). Furthermore, based on the molecular modeling of bomidin and membrane lipids, we elucidated the possible mechanism by which bomidin disrupts bacterial and viral membranes. Thus, we obtained a novel AMP with an optimized, efficient heterologous expression system for potential therapeutic application against a wide range of life-threatening pathogens.

A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides.

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Reduction of Oxidative Stress through Activating the Nrf2 mediated HO-1 Antioxidant Efficacy Signaling Pathway by MS15, an Antimicrobial Peptide from Bacillus velezensis.

The efficient culture and purification of antimicrobial peptides (AMPs), along with intense antioxidant activity, have drawn the interest to study antioxidant activity mechanism. We report the culture conditions optimization, efficient biosynthesis, and purification of an antioxidant peptide MS15 from Bacillus velezensis obtained from fermented food that would generate heme oxygenase-1 (HO-1) expression and lead to nuclear factor erythroid 2-related factor-2 (Nrf2) nuclear translocation. We explored the ability of kinetics and potency for the bacterial killing to work against various pathogenic bacteria. A bioassay showed the lysis zone of MS15 by tricine SDS-PAGE near at 6 kDa. MALDI-TOF/MS verified molecular weight, and the existence of a molecular mass of 6091 Da was reported by purity. The MIC of MS15 ranged from 2.5–160 μg/mL for many pathogenic bacteria, showing greater potency. In macrophage RAW 264.7 cells, MS15 was exposed to assess its inhibitory effect against the generation of reactive oxygen species (ROS) in oxidative stress. In the sample treated group, the translation, and transcriptional levels of CAT (catalase), GPx (glutathione peroxidase), and SOD (superoxide dismutase) were significantly greater. In short, MS15 has significant antioxidant properties, reducing ROS production in RAW 264.7 cells, and raising the translation and transcriptional rates of antioxidant enzymes with stimulating HO-1 induction facilitated by Nrf2.

Production of a Recombinant Peptide (Lasioglossin LL ΙΙΙ) and Assessment of Antibacterial and Antioxidant Activity

The use of antimicrobial peptides (AMPs) as biopreservative to replace chemical preservatives has become of interest among consumers. Current approaches of production and purification of AMPs are time-consuming and costly and may kill the producing host cells. In this study a recombinant peptide was patterned from Lasioglossin LL ΙΙΙ which is found naturally in the venom of eusocial bee, this recombinant peptide was produced to inhibit food microbial degradation with the least possible costs. The synthesis of this peptide in the periplasmic space of Escherichia coli, has facilitated the production and purification of this additive. The pET based expression system for production of a recombinant form of Lasioglossin LL ΙΙΙ using E. coli as a host was developed. The antibacterial and antioxidant activities of recombinant peptide were determined. A C-terminal poly histidine tag was used to facilitate purification by standard methods using Ni2+ affinity chromatography. The average yield of the recombinant peptide was 0.7 g/L. The findings showed great antimicrobial function of the peptide. The antioxidant activity of the peptide was lower than Butylated hydroxytoluene (BHT) synthesized antioxidant as a common antioxidant but the antioxidant potential of the peptide along with the antimicrobial activity against foodborne pathogens have shown promising benefits of the peptide. These results demonstrated that pET expression system is appropriate for the rapid and simple isolation of recombinant lasioglossin LL ΙΙΙ from E. coli and the recombinant peptide could be used as a natural preservative source. However, further studies need to be carried out to check the effect of this peptide on more microorganisms.

Heterologous expression of a broad-spectrum chimeric antimicrobial peptide in Lactococcus lactis: Its safety and molecular modeling evaluation

Over the last decade, global increase in antibiotic consumption is a major concern in the word. Antimicrobial peptides (AMPs) known as potential alternative and were considered as a safe antimicrobial agent. However, current approaches for production and purification of AMPs are costly and time-consuming. Here we show that heterologous expression of a chimeric peptide was successfully developed in Lactococcus lactis as a safe and cost-effective recombinant protein expression platform. Minimum inhibitory concentrations (MICs) of His-tag purified peptide was determined against a broad spectrum of human pathogenic bacteria consistence of Gram-positive, Gram-negative and resistance strains in deferent range from 7.24 ± 0.4 to 156.24 ± 3.0 μg/mL. Furthermore, our results showed that the peptide was not toxic to HEK and HeLa cells and even at concentrations as high as 250 μg/mL exhibited minimal hemolysis against RBCs. Additional characteristics such as thermal, protease and 50% human plasma stability were determined for cLFchimera. Molecular modeling analysis demonstrated that fusion of His-tag to the C-terminal of chimeric peptide increased peptide stability during 10 ns simulation in water. Overall, the chimeric peptide has a considerable antibacterial activity with low hemolysis, low or none in toxicity and good temperature resistance and also high stability in serum. We anticipate the established expression system could be developed and used more effectively in probiotic strains in future studies.

Recombinant production of a chimeric antimicrobial peptide in E. coli and assessment of its activity against some avian clinically isolated pathogens

Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro. A carboxy-terminal polyhistidine tag was used for purification by Ni2+ affinity chromatography with an average yield of 0.42 g/L. The His-tagged chimeric peptide showed different range of antimicrobial activity against clinically isolated avian pathogens with low chicken blood hemolysis activity and high serum stability. Overall, the results of this investigation showed the recombinant chimeric peptide was successfully expressed in pET-based expression system and could be considered as a proper alternative for some currently used antibiotics in poultry industry and drugs veterinary medicine.

Isolation and Purification of Antimicrobial Peptides from the Blood of some Animals and the Study of its Antimicrobial Activity Against some Multidrug Resistant Pathogenic Bacteria

Isolation and Purification of Antimicrobial Peptides from the Blood of some Animals and the Study of its Antimicrobial Activity Against some Multidrug Resistant Pathogenic Bacteria

참담치(Mytilus coruscus) 유래 항균 펩타이드 분리 및 정제

참담치(Mytilus coruscus) 유래 항균 펩타이드 분리 및 정제

Production of an antimicrobial peptide AN5-1 in Escherichia coli and its dual mechanisms against bacteria.

AN5-1 (YSKSLPLSVLNP) is an antimicrobial peptide isolated from the fermentation broth of Paenibacillus alvei strain AN5 (J Ind Microb Biotechnol 2013; 40: 571-9). In this study, we report the application of ubiquitin fusion technology to the expression and purification of AN5-1. Minimum inhibitory concentration (MIC) and measurement of hemolytic activity (MHC) were measured to confirm the biological activities of the expressed AN5-1. Bacterial cell membrane permeabilization was investigated to show the interaction between the AN5-1 and the bacterial cytoplasmic membrane. Furthermore, intracellular activities of the AN5-1 were determined by genomic DNA interaction assays. The results revealed AN5-1 damaging bacterial membranes and binding to bacterial genomic DNA to inhibit cellular functions, suggesting that it has multiple intracellular targets in bacteria. The application of ubiquitin fusion technology may be an excellent approach for industrial production to the expression and purification of antimicrobial peptide. Furthermore, AN5-1 was demonstrated as an antimicrobial peptide with great potentials, as bacterial resistance to AN5-1 would be not expected, due to the dual mechanisms of AN5-1 against bacteria.

Screening of serine protease inhibitors with antimicrobial activity using iron oxide nanoparticles functionalized with dextran conjugated trypsin and in silico analyses of bacterial serine protease inhibition.

Plants produce a variety of proteins and peptides which are involved in their defense against pathogens. Serine protease inhibitors are a well-established class of inhibitors correlated with plant defense. Increased levels of protease inhibitors delay cell damage and expand the cell's life-span. Recently, the rapid emergence of antibiotic-resistant microbial pathogens has prompted immense interest in purifying novel antimicrobial proteins or peptides from plant sources. Usually, the purification of protease inhibitors is accomplished by salt-extraction, ultrafiltration and affinity chromatography. Here, we developed a novel approach based on iron oxide nanoparticles conjugated to dextran functionalized with trypsin beads that accelerate the quick screening and purification of antimicrobial peptides with serine protease inhibitor activity. The method described here also works for screening other inhibitors using particular protein kinases, and it is therefore a novel tool for use as the leading method in the development of novel antimicrobial agents with protease inhibitory activity. Finally, and no less important, molecular modelling and dynamics studies of a homologous inhibitor studied here with Escherichia coli trypsin and chymotrypsin are provided in order to shed some light on inhibitor-enzyme interactions.

Purification of antimicrobial peptide from Antarctic Krill (Euphausia superba) and its function mechanism

The preliminary purification and antimicrobial mechanism of antimicrobial peptide from Antarctic Krill were studied in this paper. The results showed that the molecular weight range of antimicrobial polypeptide (CMCC-1) obtained by cation exchange chromatography was between 245-709D as detected by molecular sieve chromatography, and the minimum inhibition concentration (MIC) of CMCC-1 against Staphylococcus aureus was 5.0 mg mL−1. The antimicrobial mechanism of CMCC-1 was studied with S. aureus as indicator bacterium. Compared with control group, the results of the experimental group in which S. aureus was treated with CMCC-1 were as follows: 1) CMCC-1 could inhibit cell division at logarithmic phase. 2) The protein and reducing sugar content, and the conductivity of culture medium increased, and the activity of alkaline phosphatase and β-galactosidase could be detected in the culture medium. 3) Observation under scanning electron microscope revealed that somatic morphology became irregular, and then somatic surface became coarse. The cell became much smaller, and most somatic cells gathered. The boundary between cells became dim and finally fused as a whole. 4) Observation under transmission electron microscope showed that the surface of S. aureus became rough and the reproducing ability was restrained. The cell wall became thin and the cytoplasm shrunk. Substances inside cell leaked out, which caused cells death. 5) SDS-PAGE analysis showed that some bands disappeared, and the residual bands became vague. 6) The genomic DNA electrophoresis results showed that the genomic DNA bands of S. aureus were not degraded but the brightness significantly reduced. Thus, it is supposed that CMCC-1 could destroy the cell wall and membrane of S. aureu, increase the cell membrane permeability and the leaking-out of intracellular substances, and thus cause the death of S. aureu.

CDNA cloning, characterisation, expression and purification of antimicrobial peptides (penaeidin and anti-lipopolysaccharide factor) from Penaeus semisulcatus

cDNA cloning, characterisation, expression and purification of antimicrobial peptides (penaeidin and anti-lipopolysaccharide factor) from Penaeus semisulcatus

Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112

The antibacterial activity of antimicrobial peptides produced by strain LFB 112 was determined using the cup-plate method and a standard curve of penicillin. An effective method to evaluate the antimicrobial activity of antibacterial peptides was established. Three active fractions, Fraction A (5.0–5.5 kDa), Fraction B (3.0–3.5 kDa) and Fraction C (400–800 Da), were separated by anion-exchange, reversed phase liquid chromatography (RPLC) and gel filtration chromatography. Fraction A contained an antimicrobial peptide; its partial sequence (LVQSPNGNFAASFVLDGTK), determined by liquid chromatography-mass spectrometry (LC-MS), was homologous with antimicrobial peptide LCI. In addition, there were three major ingredients in fraction B (3,371.811, 3,442.379 and 3,486.596 Da, respectively), and several small peptides (400–800 Da) in fraction C. After purification, the potencies of fractions A, B and C were 188.6, 11,217.26 and 10,290.63 U/mg, respectively. Furthermore, antimicrobial peptides in fraction B, apparently different from known antimicrobial peptides produced by Bacillus subtilis (based on molecular weight), and small (molecular weight 400–800 Da) antimicrobial peptides produced by B. subtilis LFB112 were also identified. Antimicrobial peptides produced by strain LFB112 with high potency against several undesirable bacteria were identified. These compounds have potential as alternatives to known antibiotics.

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.