Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag

Wright, Oliver; Yoshimi, Tatsuya; Tunnacliffe, Alan

doi:10.1016/j.nbt.2011.11.001

Cited by 10 publications

(7 citation statements)

References 24 publications

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“…To test whether C. difficile VM can bind IVA, we coproduced His 6 -tagged C. difficile VM (VM Cd ) with hemagglutinin (HA)-tagged C. difficile IVA in E. coli and measured the ability of HA-tagged IVA to copurify with His 6 -tagged VM Cd using Ni 2+ -affinity chromatography. To facilitate the production and detection of VM Cd , we fused it to CPD-His 6 , an inducible, self-cleaving protease domain ( 42 ) that can promote the production and purification of small amphipathic helices ( 43 , 44 ). As a positive control, we measured the ability of HA-tagged B. subtilis IVA to copurify with B. subtilis VM-CPD-His 6 .…”

Section: Resultsmentioning

confidence: 99%

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

Ribis

Ravichandran

Putnam

et al. 2017

mSphere

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The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.

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Section: Resultsmentioning

confidence: 99%

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

Ribis

Ravichandran

Putnam

et al. 2017

mSphere

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“…Its presence was detected in various cells and tissues such as circulating neutrophils, myeloid bone marrow cells, epithelial cells of the skin, and tissues in the gastrointestinal tract, mouth, esophagus, and lungs . Recombinant production of LL‐37 in E. coli and Pichia pastoris have been reported previously . Regarding plant systems, the gene coding for cathelicidin LL‐37 or its variant was integrated into genome of Chinese cabbage ( Brassica rapa var.…”

Section: Introductionmentioning

confidence: 99%

Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37

et al. 2018

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The peptide LL-37, a component of the human innate immune system, represents a promising drug candidate. In particular, the development of low-cost production platform technology is a critical bottleneck in its use in medicine. In the present study, a viable approach for the LL-37 production in transgenic barley is developed. First, comparative analyses of the effects of different fused peptide epitope tags applicable for accumulation and purification on LL-37 production yield are performed using transient expression in tobacco leaves. Following the selection of the most yielding fusion peptide strategies, eight different constructs for the expression of codon optimized chimeric LL-37 genes in transgenic barley plants are created. The expression of individual constructs is driven either by an endosperm-specific promoter of the barley B1 hordein gene or by the maize ubiquitin promoter. The transgenes are stably integrated into the barley genome and inherited in the subsequent generation. All transgenic lines show normal phenotypes and are fertile. LL-37 accumulated in the barley seeds up to 0.55 mg per 1 kg of grain. The fused epitope tags are cleaved off by the use of enterokinase. Furthermore, in planta produced LL-37 including the fused versions is biologically active.

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“…However, a cost‐effective and scalable method of active production is required in order to commercialize the bacterial peptides. Bacterial expression of heterologs proteins is an easy and inexpensive tool for producing large amounts of recombinant proteins 18. Production of recombinant expression of AMPs in Escherichia coli is potentially limited because of their toxicity to host cells and susceptibility to proteolytic degradation, which can be avoided using fusion protein approach 19.…”

Section: Introductionmentioning

confidence: 99%

“…20 However, the very few works describing MAG2 expression in E. coli , including using fusing partners,21 may indicate that it is very difficult to produce MAG2 in bacteria. As Magainin displays high antibacterial and antitumor activities,11, 12 the development of a method to obtain recombinant and functional Magainin but at low cost is crucial for its expansion as a therapeutic agent 18, 19. Moreover, potent analogs of MAG2 have been obtained by substitutions of glycine or serine with alanine residues and amidation of the C‐terminus, which resulted in enhanced α‐helical structure and antimicrobial activity.…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression and purification of the antimicrobial peptide magainin‐2

Ramos

Moreira

Rodrigues

et al. 2012

Biotechnology Progress

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Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.

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Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag

Cited by 10 publications

References 24 publications

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37

Recombinant expression and purification of the antimicrobial peptide magainin‐2

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