Simple purification of human antimicrobial peptide dermcidin (MDCD-1L) by intein-mediated expression in E.coli

Hong, In-Pyo; Kim, Yong-Seok; Choi, Shin-Geon

doi:10.4014/jmb.0907.07029

Cited by 13 publications

(6 citation statements)

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“…Next, we produced and purified large amounts of the full-length Y-P30/dermcidin GFP- tagged precursor containing the signal peptide using the Intein expression system [ 25 ]. We applied the purified protein at 10 μg/ml medium to differentiated primary cortical neurons, and we found the GFP-fluorescence aggregating at dendritic sites and a protrusions that resemble spines.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Y-P30/Dermcidin expression and properties of the Y-P30 peptide

et al. 2014

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BackgroundThe survival promoting peptide Y-P30 has a variety of neuritogenic and neuroprotective effects in vitro and in vivo. In previous work we reported the expression of Y-P30/dermcidin in maternal peripheral blood mononuclear cells (PBMCs) and the transport of the protein to the fetal brain. In this study we analyzed hormonal regulation of Y-P30 in human immune cells and expression of Y-P30 in the placenta. We further studied the stability and secretion of the Y-P30 peptide.ResultsWe found indications that Y-P30 might be produced in human placenta. The Y-P30 mRNA was rarely found in isolated human PBMCs and alpha-feto-protein, human chorionic gonadotropin as well as estradiol combined with progesterone could not induce Y-P30 expression. Y-P30 was found to be extraordinarily stable; therefore, contamination with the peptide and the Y-P30/Dermcidin precursor mRNA is a serious concern in experiments looking at the expression of Y-P30/Dermcidin. In cultured cell lines and primary neurons we found that Y-P30 could be released, but neuronal uptake of Y-P30 was not observed.ConclusionsOur data suggest that a source of Y-P30 apart from eccrine glands might be the placenta. The peptide can be secreted together with the signaling peptide and it might reach the fetal brain where it can exert its neuritogenic functions by binding to neuronal membranes.

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Section: Resultsmentioning

confidence: 99%

“…For bacterial expression, Y-P30 containing the signal peptide (aa 1–49) was subcloned into a pTYB21 vector. Expression and purification of Y-P30 via the Intein system was performed according to manufacturer’s instructions (Impact kit, NEB) and previously published protocols [ 25 ].…”

Section: Methodsmentioning

confidence: 99%

Analysis of Y-P30/Dermcidin expression and properties of the Y-P30 peptide

et al. 2014

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“…Cattle sweat has high contents of total protein nitrogen and the inorganic salt and relatively high urea content [35]. The human DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations [36]. The antimicrobial activity of DCD-1L is maintained over a broad pH range and at high salt concentrations that resemble the conditions in human sweat [17].…”

Section: Discussionmentioning

confidence: 99%

Characterization and Antimicrobial Efficacy of Bovine Dermcidin, a Novel Antimicrobial Peptide Gene

Sabry¹,

Moussa²

2019

Preprint

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Description of a novel bovine antimicrobial peptide and its antimicrobial spectrum. RNA isolation and RT-PCR were done from various tissues. DCD peptide was synthesized, and antimicrobial activity was analyzed. Bovine dermcidin gene contains five exons intermittent by 4 introns. Bovine DCD-mRNA was 398 bp with ORF 336 bp. Bovine DCD was expressed in skin and blood. Analysis of the amino acid compositions revealed that cysteine was repeated 6 times indicating the presence of 3 disulfide bonds that play role in the peptide stability. Staphylococcus epidermidis, Streptococcus bovis, and Enterococcus faecalis were affected by Bovine DCD peptide. Highest antimicrobial effect was at 50 and 100 µg/ml. The effect on Escherichia coli and Candida albicans was slightly low. In all bacteria, Bovine DCD peptide activity did not affect by varying pH values, but in Staphylococcus aureus, the activity was affected greatly at pH 4.5 and 5.5. The optimum salt concentrations were 100 and 50 mM NaCl with all bacterial strains and E. coli, respectively. In case of C. albicans, the antimicrobial activity of Bovine DCD peptide decreased with increasing the pH value regardless the NaCl concentration. The pH 6.5 of the sweat buffer was the optimum for the Bovine DCD peptide activity.

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“…In another study, recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads (Hong et al, 2010). Intein system eliminates the need for exogenous proteases or chemicals that are usually required for tag removal and allows the target peptide to be purified through relatively simple procedures.…”

Section: Discussionmentioning

confidence: 99%

SUMO-based expression and purification of dermcidin-derived DCD-1L, a human antimicrobial peptide, inEscherichia coli

Mohanraj

Kinnunen

Kaya

et al. 2018

Preprint

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The dermcidin-derived peptide DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. Thus, it offers a promising alternative to conventional antibiotics. Furthermore, it plays a role in wound healing, atopic dermatitis and acne vulgaris, indicating applications in cosmetic industries. Recently, dermcidin has been identified as a tumor marker improving cancer prognosis. Hence, large quantities of purified DCD-1L peptide are required to meet the needs of basic research and clinical trials. In the current study, we demonstrate SUMO-based heterologous DCD-1L production in Escherichia coli, followed by affinity chromatography purification. The SUMO tag is cleaved with SUMOspecific protease following purification, leaving free DCD-1L peptide without any additional amino acids. The mass of the peptide was further confirmed by MALDI-TOF-TOF analysis. Furthermore, the cleaved DCD-1L showed antimicrobial activity against the E. coli DH5 alpha test strain. The production and purification of DCD-1L using SUMO tag compare advantageously to other protocols previously described. Thus, the SUMO tag system enables large scale recombinant production of the antimicrobial peptide DCD-1L, which constitutes pharmaceutical and therapeutic potential as an alternative antibiotic.

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Simple purification of human antimicrobial peptide dermcidin (MDCD-1L) by intein-mediated expression in E.coli

Cited by 13 publications

References 0 publications

Analysis of Y-P30/Dermcidin expression and properties of the Y-P30 peptide

Analysis of Y-P30/Dermcidin expression and properties of the Y-P30 peptide

Characterization and Antimicrobial Efficacy of Bovine Dermcidin, a Novel Antimicrobial Peptide Gene

SUMO-based expression and purification of dermcidin-derived DCD-1L, a human antimicrobial peptide, inEscherichia coli

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