scholarly journals Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7142
Author(s):  
Junhao Cheng ◽  
Marhaba Ahmat ◽  
Henan Guo ◽  
Xubiao Wei ◽  
Lulu Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Bacterial Infections ◽  
Expression System ◽  
Enterotoxigenic Escherichia Coli ◽  
Proteinase K ◽  
Hybrid Peptide ◽  
Tev Protease ◽  
High Level

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

scholarly journals Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tessa B. Moyer ◽  
Ashleigh L. Purvis ◽  
Andrew J. Wommack ◽  
Leslie M. Hicks
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
Gram Negative Bacteria ◽  
Amaranthus Tricolor ◽  
Core Motif ◽  
Gram Negative ◽  
E Coli ◽  
Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

scholarly journals High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme

1993 ◽  
Vol 294 (1) ◽  
pp. 69-77 ◽  
Author(s):  
C Wang ◽  
M R Lee
Keyword(s):  
Escherichia Coli ◽  
Dissociation Constants ◽  
Expression System ◽  
Deae Cellulose ◽  
Basic Amino Acid ◽  
Coated Vesicle ◽  
Amino Acid Residues ◽  
High Level ◽  
Binding Component

We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.

scholarly journals Casein-Derived Antimicrobial Peptides Generated by Lactobacillus acidophilus DPC6026

2006 ◽  
Vol 72 (3) ◽  
pp. 2260-2264 ◽  
Author(s):  
M. Hayes ◽  
R. P. Ross ◽  
G. F. Fitzgerald ◽  
C. Hill ◽  
C. Stanton
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Lactobacillus Acidophilus ◽  
Sodium Caseinate ◽  
Enterobacter Sakazakii ◽  
Potential Use

ABSTRACT Three peptides produced by a Lactobacillus acidophilus DPC6026 fermentation of sodium caseinate and showing antibacterial activity against pathogenic strains Enterobacter sakazakii ATCC 12868 and Escherichia coli DPC5063 were characterized. These peptides were all generated from bovine αs1-casein and identified as IKHQGLPQE, VLNENLLR, and SDIPNPIGSENSEK. These peptides may have bioprotective applicability and potential use in milk-based formula, which has been linked to E. sakazakii infection in neonates.

Mycoses, bacterial infections and antibacterial activity in sandifies (Psychodidae) and their possible role in the transmission of leishmaniasis

Parasitology ◽  
1985 ◽  
Vol 90 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Y. Schlein ◽  
I. Polacheck ◽  
B. Yuval
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacterial Infections ◽  
Leishmania Major ◽  
Bacterial Species ◽  
Jordan Valley ◽  
High Incidence ◽  
Group A

High incidence of mycoses were found in the guts and malpighian tubes of Phlebokomus papatasi from the Jordan Valley and P. tobbi from Zakinthos, Greece. Infections with several different bacteria were also found in the guts of female P. tobbi. Fungi cultured from guts of laboratory reared P. papatasi that had similar mycoses were identified as Aspergillus scierotiorum and Saccharomyces cerevisiae. Fungi-infected laboratory reared P. papatasi were refractory to artificial infections with a Leishmania major strain specific to them. The crop contents of P. papatasi, where sugar meals are stored, demonstrated antibacterial activity against the following bacterial species in culture: Escherichia coli, Staphylococcus aureus, Shigella sonnei, Streptococcus group A and Pseudomonas aeruginosa. It is postulated that the bacteria-free gut normal to sandflies is effected by the bacterial inhibitor present in the crop.

scholarly journals Isolation of antibiotics producing bacteria from marine soil and comparative analysis of same with commercially available drugs

2021 ◽  
Vol 16 (1) ◽  
pp. 070-076
Author(s):  
Chinyelu Nkiru Umeaku ◽  
Chisom Faith Ohagwam ◽  
Chiamaka Ijeoma Chris-Umeaku
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Comparative Analysis ◽  
Bacterial Infections ◽  
Streptomyces Griseus ◽  
Zone Of Inhibition ◽  
Preliminary Screening ◽  
Port Harcourt ◽  
Marine Soil

The isolation of antibiotic producing bacteria from marine soil and comparative analysis of same with ciprofloxacin and amoxicillin against staphylococcus aureus and Escherichia coli was carried out in a Microbiology Laboratory of Chukwuemeka Odumegwu Ojukwu University, Uli. This was done to isolate antibiotic producing bacteria and compare same with existing commercially available antibiotics with a view to using marine soil in the treatment of common bacterial infections. Soil samples were collected from Bonny Island Sea, Port Harcourt. One gram of mixed soil sample was serially diluted and spread-plated on nutrient agar plates. The representative isolates obtained were sub-cultured to get a pure culture. Morphological, biochemical, physiological characteristics of the bacteria were analyzed. Agar well diffusion was carried out. One isolate had a substantial antibacterial activity with 3.5mm zone of inhibition against two test bacteria used in the preliminary screening. The isolate was marked as Streptomyces (STR I) and was identified as Streptomyces griseus while other isolates did not show any antibacterial activity. Ciprofloxacin showed the highest antibacterial activity to both Staphylococcus aureus and Escherichia coli of 3.7mm and 4.0mm respectively while Amoxicillin showed antibacterial activity of 3.5mm and 2.7mm respectively. This reveals that antibiotic producing bacteria from marine soil are also effective in antimicrobial activity and could be used for antimicrobial chemotherapy.

scholarly journals Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Syed Shoaib Ahmed ◽  
Mohsina Akhter ◽  
Muhammad Sajjad ◽  
Roquyya Gul ◽  
Sana Khurshid
Keyword(s):  
Escherichia Coli ◽  
Clostridium Thermocellum ◽  
Expression System ◽  
Alpha Helix ◽  
Cellular Protein ◽  
Industrial Applications ◽  
Heat Inactivation ◽  
Potential Candidate ◽  
Cellulolytic Activity ◽  
High Level

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum (C. thermocellum) by cloning in an Escherichia coli (E. coli) expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a Km of 19.83 mM and a Vmax of 0.12 μmol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C. thermocellum a potential candidate in industrial applications.

scholarly journals Development of technology of disinfectants based on soda production waste

2019 ◽  
Vol 140 ◽  
pp. 01004
Author(s):  
Elina Salmanova ◽  
Lyasan Araslanova ◽  
Iren Tuktarova ◽  
Alexey Nazarov
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Candida Albicans ◽  
Iodine Monochloride ◽  
Waste Production ◽  
Bacillus Subtilus ◽  
High Level ◽  
Technical And Economic Indicators

The main requirements for the development of fundamentally new disinfectants with a high level of efficiency are to reduce toxicity by increasing the technical and economic indicators in the technology of their application. In this paper, an innovative composition of a disinfectant based on iodine monochloride in the complex of waste production of soda and polyvinylpyrrolidone is proposed. The dependence of stability and antibacterial activity of the developed disinfectant is studied. High indicators of stability (3 years) and antibacterial activity were obtained on the example of Staphylococcus aureus (S. aureus), Enterococcus fecalis (E. fecalis), Pseudomonas aeruginosa (P. aerginosa), Bacillus subtilus (B. Subtilis No. 12) at the concentration 0.027-0.055, DS versus commercial drug Zhavilar. The antibacterial activity of the disinfectant DS, DS-1 is maintained up to 0.0044-0.0088 for Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Escherichia coli and exceeds the efficiency of the commercial drug Betadine by 8-28 times. Thus, the developed formulations are highly effective, while low-toxic and safe for the environment and humans.

scholarly journals The Ethnobotanical Survey, Antibacterial Activity and Phytochemical Screening of Extracts of Prosopis africana (Guill. & Perr.) Taub

2020 ◽  
pp. 39-47
Author(s):  
Bance Alimata ◽  
Magnini René Dofini ◽  
Compaore Souleymane ◽  
Compaore Eli ◽  
Ouedraogo Noufou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Bacterial Infections ◽  
Traditional Healers ◽  
Diffusion Method ◽  
Phytochemical Screening ◽  
Ethnobotanical Survey ◽  
Prosopis Africana ◽  
Phytochemical Constituents ◽  
Ethnomedicinal Uses

Aims: To investigate the ethnomedicinal uses of Prosopis africana (Guill. & Perr.) Taub and to screen the antimicrobial property as well as determine the phytochemical constituents of leaves, stems and root bark. Study Design: Ethnobotanical surveys, antibacterial activity and phytochemical screening of extracts of P. africana. Place and Duration of Study: The ethnobotanical survey was conducted during June 2015 in Zounweogo Province. The experiments were conducted at the Department of Medicine and Traditional Pharmacopeia-Pharmacy (MEPHATRA-PH) of the Institute of Research in Health and Laboratory of Applied Biochemistry and Chemistry (LA.BIO.C.A), University Joseph KI-ZERBO. Methodology: The semi-structured questionnaires were administrated to 36 traditional healers and elucidated out on the ethnomedicinal uses of P. africana in treating bacterial infections, the plant parts used and the mode of administration. The antimicrobial activity of different polar extracts of the leaves, the stem and root were evaluated by using the agar diffusion method and the determination of the minimal inhibitory concentration (MIC) of extracts via microdilution method. The phytochemical constituents of all extracts were also screened air Ciulei method. Results: The traditional healers consisted of 64% women and 36% men were surveyed.                 P. africana is used to treat tooth decay, childhood diarrhoea and chronic wounds. Leaves and the stem bark are the most commonly used plant part in treating bacterial infections while the roots are primarily used for other therapeutic purposes. The main method of administration was decoction. Methanol extracts of the leaves showed better antibacterial activity on all bacterial strains than aqueous extracts: Escherichia coli ATCC 25922 (MIC = 390 µg/ml; diameter of inhibition = 13.00 ±1.00 mm), Staphylococcus aureus ATCC 25923 (MIC = 390 µg/ml; diameter of inhibition = 12.33 ± 1.53 mm), Escherichia coli ATCC 35218 (MIC = 3120 µg/ml; diameter of inhibition = 13±1,00 mm), Pseudomonas aeruginosa PAO1 (MIC = 12500 µg/ml; diameter of inhibition = 12.33±0.58 mm). Alkaloid salts, tannins, sterols and triterpenes, saponosides, flavonic glycosides and leucoanthocyans were found in extracts of the leaves, as well as in the barks of the stem and root. Conclusion: These results demonstrated that P. africana is a potent source of antimicrobial compounds and could justify its traditional use of in the folklore medicine of Zounweogo Province.

Sign in / Sign up

Export Citation Format

Share Document