High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

Krahulec, Ján; Hyršová, Marcela; Pepeliaev, Stanislav; Jílková, Jana; Černý, Zbyněk; Machálková, Jana

doi:10.1007/s00253-010-2736-7

Cited by 37 publications

(17 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…There were many attempts to express cathelicidin in different expression systems using fusion partners such as: thioredoxin, small ubiquitin-related modifier (SUMO), glutathione transferase (GST) and family III cellulose-binding module (CBM) [22,[96][97][98][99][100][101][102]].…”

Section: Expressionmentioning

confidence: 99%

Unique features of human cathelicidinLL‐37

et al. 2015

View full text Add to dashboard Cite

Cathelicidins are antimicrobial peptides produced by humans and animals in response to various pathogenic microbes. This review intends to provide a brief overview of the expression, structure, properties and function of human cathelicidin LL-37 which may be a therapeutic agent against a variety of bacterial and viral diseases, cancers, and hard-to-heal wounds. Cathelicidins act as a primary defense against bacteria and other pathogens in the case of inflammation. They are able to kill bacteria and fungi, inhibit and destroy bacterial biofilms, and possess antiviral and antiparasitics properties. They can also play a role in angiogenesis, wound healing, and the regulation of apoptosis. The host defense peptide LL-37 has emerged as a novel modulator of tumor growth and metastasis in carcinogenesis of various types of cancers. LL-37 is an antimicrobial peptide able of inducing various effects. It acts as an anti- and pro- inflammatory factor. Cathelicidins are able to directly and selectively destroy membranes of various microbes and cancer cells, but they do not attack normal cells. The role of cathelicidins in cancer is double-sided. They play an important role in killing cancer cells and may provide a new possibility for the development of cancer therapeutics. However, they also can participate in carcinogenesis. Due to its activity spectrum LL-37 could be applied in pharmacotherapy. Cathelicidin peptides could serve as a template for the development of modern anti-microbial and anti-viral drugs. LL-37 is an excellent candidate to develop into therapeutics for infected wounds.

show abstract

Section: Expressionmentioning

confidence: 99%

Unique features of human cathelicidinLL‐37

et al. 2015

View full text Add to dashboard Cite

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

“…One group recently reported that the expression system based on T7 polymerase under the control of lac promoter and thioredoxin as the fusion partners allowed almost six times higher yield than that from our system [Krahulec et al, 2010]. Another recent article reported direct expression of LL-37 in Pichia pastoris, and the band corresponding to LL-37 was barely visible on the gel [Hong et al, 2006], indicating a very low expression.…”

Section: Discussionmentioning

confidence: 75%

Characterization of factors favoring the expression and purification of recombinant LL-37 from Escherichia coli

Moon

Kang

Cho

et al. 2011

J Korean Soc Appl Biol Chem

View full text Add to dashboard Cite

The only human antimicrobial peptide, LL-37, was overexpressed in soluble form using Escherichia coli-based expression system. Recombinant LL-37 production in E. coli was optimized for use in large quantities for studying antimicrobial activity against Helicobacter pylori strains. We previously reported a method to express and purify LL-37 using Glutathione S-transferase (GST) fusion system. Herein presents method suitable for producing recombinant LL-37 from the recombinant GST-LL-37 fusion protein by recovering from both soluble protein fractions and inclusion bodies. Compared to the yield reported previously, the yield of recombinant GST-LL-37 protein was much improved to 10 mg/L of culture by affinity chromatography using GSTrap FF (1 mL) affinity chromatography column. These results suggest that the production method used in present study is useful in obtaining large quantity of recombinant LL-37 peptide. The optimized recovery protocol from inclusion bodies was highly contributable in raising yield of GST-LL-37 fusion protein. When treated with Factor Xa, GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies released an approximate 4.5-kDa protein, which was the expected size of LL-37; GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies was also confirmed by enzymatic digestion with thrombin, which produced LL-37 peptide containing six extra amino acid residues, Gly-Ile-Ile-Glu-Gly-Arg, on Nterminus of LL-37. Purified recombinant LL-37 showed nearly identical antimicrobial activities against H. pylori strains as that of synthetic LL-37, suggesting its application to functional study with therapeutic potential on gastric pathogen.

show abstract

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

Cited by 37 publications

References 29 publications

Unique features of human cathelicidinLL‐37

Unique features of human cathelicidinLL‐37

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

Characterization of factors favoring the expression and purification of recombinant LL-37 from Escherichia coli

Contact Info

Product

Resources

About