Recombinant production of antimicrobial peptides in Escherichia coli: A review

Li, Yifeng

doi:10.1016/j.pep.2011.08.001

Cited by 261 publications

(202 citation statements)

References 90 publications

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“…It is well known that the expression of cysteine-rich AMPs such as snakin peptides in E. coli cells is a significant challenge because the formation of disulfide bridges in the expressed protein is inefficient, leading to incorrect folding and destabilization of the tertiary structure [40,41]. These limitations of the E. coli expression system prompted us to select the methylotrophic P. pastoris strain in order to develop a method for the large-scale preparation of a disulfide-rich SN-1 containing about 20% cysteine in its amino acid sequence.…”

Section: Hemolytic Assays Of Recombinant Sn-1mentioning

confidence: 99%

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Kuddus

Rumi

Tsutsumi

et al. 2016

Protein Expression and Purification

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Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum).Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris.A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and 1 H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active 3 SN-1 in P. pastoris.4

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Section: Hemolytic Assays Of Recombinant Sn-1mentioning

confidence: 99%

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Kuddus

Rumi

Tsutsumi

et al. 2016

Protein Expression and Purification

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“…Nevertheless, there are also some limitations. Factors that influence the expression level of the target peptide include inhibition of host growth by AMPs natural activities, AMP instability against bacterial and yeast proteases, and the inability to perform appropriate post-translational modifications (such as disulphide bonds) and produce recombinant proteins in the form of inactive inclusion bodies [14,15]. Furthermore, due to the requirement for fermentation, the large-scale production of AMPs in bacteria and yeast systems is limited, and the expensive downstream process significantly increases the production costs [16,17].…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

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Antimicrobial peptides (AMPs) with their broad and selective antimicrobial activity and lowered risk of resistance development in microbial populations are promining as a new alternative candidate to conventional antibiotics. Plant-based expression systems can be employed as cost-effective and high scale-up capacity production hosts for recombinant expression of AMPs. LFchimera is a chimerical peptide containing LFcin and LFampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Here, the LFchimera sequence was codon-optimized for tobacco and fused with endoplasmic reticulum retention signals along with a CaMV 35S promoter and then transferred by agrobacterium-mediated transformation. The integration and expression of the transgene in tobacco plants were confirmed by polymerase chain reaction (PCR) and RT-PCR, respectively. Recombinant production of the peptide was confirmed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), and peptide functional activity was confirmed by the antibacterial evaluation of total protein extracts against various clinical and phytopathogenic bacteria. Finally, LFchimera peptide was partially purified using an affinity column, and the antibacterial activity was shown. Taken together, these results confirmed that tobacco can be utilized for the recombinant production of antimicrobial peptides such as LFchimera.

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“…Unfortunately, although they could be used in several fields of application, including agriculture, their production costs are elevated as the most effective approach so far is by chemical synthesis. In addition, there are heterologous expression strategies using Escherichia coli as host [6]. Examples of this approach include the production of cecropin [7,8], moricin [9], human defensins [10], magainin [11,12] and concatemers, such as buforin II.…”

Section: Introductionmentioning

confidence: 99%

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide

Roldán-Tapia

Anné

Reyes³

et al. 2017

PPL

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Defense mechanisms of plants against phytopathogens include cationic antimicrobial peptides (CAPs). The broad-spectrum activity of these peptides has been evaluated previously against different phytopathogens. Their lack of toxicity for plants and animals is a promising feature for pest control. Although, some attempts have been made previously in order to increase their heterologous expressions, the employed strategies have so far proven to be ineffective. Low production yield and elevated costs are the obstacles to overcome. In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. This system contains elements that allow the increase of the efficiency of the peptide's expression. The main elements included in this cassette are the sequences of the promoter and signal peptide from a subtilisin inhibitor gene of Streptomyces venezuelae. This allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The results obtained demonstrate the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies.

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Recombinant production of antimicrobial peptides in Escherichia coli: A review

Cited by 261 publications

References 90 publications

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide

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