In vitro production and antifungal activity of peptide ABP-dHC-cecropin A

“…The antibacterial activity curves of ABP-dHC-cecropin A are shown in Figure 6B; 0. to proteolytic degradation, and low production yield. In our previous work, we used a monomer ABP-dHC-cecropin A SUMO fusion protein for expression [15]. Although the fusion protein was soluble, the quantity produced was not very large.…”

Section: Antimicrobial Activity Assaymentioning

confidence: 99%

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli

Zhang

Movahedi

Wei

et al. 2016

Analytical Biochemistry

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“…E. coli expression systems have been extensively demonstrated to effectively produce AMPs, but the AMP must be fused to a carrier protein in order to protect the bacterium from antimicrobial activity (Li, 2009). Various fusion partners have been used, such as SUMO (Li et al , 2009a; Zhang et al , 2015), GST (Liang et al , 2006) and TRX (Tian et al , 2009), but these must be removed post-production to restore antimicrobial activity to the AMP, adding an extra cost to production. A variety of ingenious methods have been proposed to perform the cleavage event without the use of proteases post-production (Tian et al , 2009; Ke et al , 2012), but, with one exception (Rothan et al , 2014), AMPs that retain antimicrobial activity while still bound to the fusion partner have not been produced in bacteria.…”

Section: Introductionmentioning

confidence: 99%

Making plants into cost-effective bioreactors for highly active antimicrobial peptides

Ghidey

Islam

Pruett

et al. 2019

Preprint

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18As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have 19 grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; 20 however, reported heterologous AMP expression is much lower in plants compared to E. coli expression systems 21 and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. We wondered if there were a 22 physical factor that made heterologous AMPs difficult to express in plants. Using a meta-analysis of protein 23 databases, we determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. 24To apply this finding to plant expression, we tested the transient expression of 10 different heterologous AMPs, 25 ranging in charge from +7 to -5, in the the tobacco, Nicotiana benthamiana. We first tested several carrier proteins 26 and were able to express AMPs only with elastin-like polypeptide (ELP). Conveniently, ELP fusion allows for a 27 simple, cost-effective temperature shift purification. Using the ELP system, all five anionic AMPs expressed well, 28with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity against Staphylococcus 29 epidermidis was an order of magnitude stronger (average MIC = 0.26 µM) than that typically seen for AMPs 30 expressed in E. coli expression systems. Unexpectedly, this high level of antimicrobial activity was associated with 31 the uncleaved fusion peptide. In contrast, all previous reports of AMPs expressed in both plant and E. coli 32 expression systems show cleavage from the fusion partner to be required before activity is seen. In summary, we 33 describe a means of expressing AMP fusions in plants in high yield, purified with a simple temperature-shift 34 protocol, resulting in a fusion peptide with high antimicrobial activity, without the need for a peptide cleavage step. 35 36 37 42 antibiotics has led to pathogenic and commensal bacteria incorporating and retaining genes for detoxification or 43 export of antibiotics, inevitably resulting in resistance to all new antibiotics introduced (Aminov et al. 2010; Thung 44 et al., 2015; Enright et al. 2002; Nathan and Cars, 2014).45 Both of these undermining factors are addressed by antimicrobial peptides (AMPs). First, the resources 46 available to develop new AMP drugs is vast and recombinant peptide variants can be quickly generated, unlike the 47 slow discovery and development cycle for antibiotics. AMPs are abundant across the taxa, being found in 48 vertebrates, insects, fungi and plants. Thousands of AMPs have been isolated and tested experimentally (Wang et 49 al., 2015) and many more can be discovered using algorithms to scan genome data bases (Islam et al., 2018b).50 Second, though resistance to AMPs has been shown to develop in bacteria (Kubicek-Sutherland et al., 2017), the 51 multiple antimicrobial activities and low affinity targets typical of AMPs have been thought to make them more 52 difficult targ...

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“…To conquer the difficulty of host self-destruction by the toxic target peptides, the fusion protein method was proposed to reduce the toxicity. This method has been used for many peptides expression, including thioredoxin [10], green fluorescent protein (GFP) [14], small ubiquitin-related modifier (SUMO) [15], and cationic elastin-like polypeptides (CELP) [16]. In this study, intein (INT) was used as the fusion peptide to reduce the toxicity of the antibacterial cecropin B2.…”

Section: Introductionmentioning

confidence: 99%

Antibacterial Peptide CecropinB2 Production via Various Host and Construct Systems

et al. 2016

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Abstract:Cecropin is a cationic antibacterial peptide composed of 35-39 residues. This peptide has been identified as possessing strong antibacterial activity and low toxicity against eukaryotic cells, and it has been claimed that some types of the cecropin family of peptides are capable of killing cancer cells. In this study, the host effect of cloning antibacterial peptide cecropinB2 was investigated. Three different host expression systems were chosen, i.e., Escherichia coli, Bacillus subtilis and Pichia pastoris. Two gene constructs, cecropinB2 (cecB2) and intein-cecropinB2 (INT-cecB2), were applied. Signal peptide and propeptide from Armigeres subalbatus were also attached to the gene construct. The results showed that the best host for cloning cecropinB2 was P. pastoris SMD1168 harboring the gene of pGAPzαC-prepro-cecB2 via Western blot confirmation. The cecropinB2 that was purified using immobilized-metal affinity chromatography resin showed strong antibacterial activity against the Gram-negative strains, including the multi-drug-resistant bacteria Acinetobacter baumannii.

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In vitro production and antifungal activity of peptide ABP-dHC-cecropin A

Cited by 12 publications

References 33 publications

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli

Making plants into cost-effective bioreactors for highly active antimicrobial peptides

Antibacterial Peptide CecropinB2 Production via Various Host and Construct Systems

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