Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies

Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of (15)N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database (), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.     

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount.     

A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation

The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.     

Antiviral Activity of the Human Cathelicidin,LL-37, and Derived Peptides on Seasonal and Pandemic Influenza A Viruses

Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties.     

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.     

Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles

As a key component of the innate immunity system, human cathelicidin LL-37 plays an essential role in protecting humans against infectious diseases. To elucidate the structural basis for its targeting bacterial membrane, we have determined the high quality structure of (13)C,(15)N-labeled LL-37 by three-dimensional triple-resonance NMR spectroscopy, because two-dimensional (1)H NMR did not provide sufficient spectral resolution. The structure of LL-37 in SDS micelles is composed of a curved amphipathic helix-bend-helix motif spanning residues 2-31 followed by a disordered C-terminal tail. The helical bend is located between residues Gly-14 and Glu-16. Similar chemical shifts and (15)N nuclear Overhauser effect (NOE) patterns of the peptide in complex with dioctanoylphosphatidylglycerol (D8PG) micelles indicate a similar structure. The aromatic rings of Phe-5, Phe-6, Phe-17, and Phe-27 of LL-37, as well as arginines, showed intermolecular NOE cross-peaks with D8PG, providing direct evidence for the association of the entire amphipathic helix with anionic lipid micelles. The structure of LL-37 serves as a model for understanding the structure and function relationship of homologous primate cathelicidins. Using synthetic peptides, we also identified the smallest antibacterial peptide KR-12 corresponding to residues 18-29 of LL-37. Importantly, KR-12 displayed a selective toxic effect on bacteria but not human cells. NMR structural analysis revealed a short three-turn amphipathic helix rich in positively charged side chains, allowing for effective competition for anionic phosphatidylglycerols in bacterial membranes. KR-12 may be a useful peptide template for developing novel antimicrobial agents of therapeutic use.     

Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli

The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria.     

The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.     

内含肽介导PHB纯化人源抗菌肽LL-37体系的构建

新颖的内含肽介导PHB纯化蛋白体系,是一种高效表达、自动切割、纯化方便、费用低廉的蛋白表达纯化体系,有利于蛋白规模化纯化。本研究选用对原核细胞具有毒害作用的小肽--人源抗菌肽LL-37作为纯化对象,通过基因工程技术,构建内含肽介导PHB纯化人源抗菌肽LL-37体系,并利用该体系纯化LL-37。研究结果表明,本研究构建的内含肽介导PHB纯化人源抗菌肽LL-37体系可高效表达LL-37融合蛋白,利用构建的纯化体系能对目的蛋白进行纯化。     

Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system

In this work, the intein fusion approach was used for expression and purification of cathelicidin-like peptide SMAP-29 from Escherichia coli cultures. To overcome the high toxicity of the antimicrobial peptide against host cells, both C- and N-terminal fusions with Sce VMA intein were evaluated. The fusion of SMAP-29 with the N-terminus of intein had a dramatic lethal effect. In contrast, chimeric constructs harboring SMAP-29 linked to the C-terminus of intein displayed no significant inhibition of bacterial growth. Expression of intein-SMAP fusion protein was then induced in ER2566 E. coli strain by IPTG addition and different experimental conditions were tested in order to optimize the recovery of the soluble protein complex. Peptide purification was carried out by affinity chromatography: the chitin binding domain linked to intein was used to immobilize the chimeric protein on a chitin column and intein-mediated splicing of target peptide was obtained by thiol addition. Microbroth dilution assay showed that recombinant SMAP-29 displayed a high, dose-dependent bactericidal activity. These data demonstrate that the fusion of SMAP-29 with C-intein was able to inactivate the antimicrobial properties of the cathelicidin peptide allowing the expression of fusion protein in the host cell. The intein-mediated purification supplied an effective way to recover the fusion partner in its proper biologically active form.     

The Human Cathelicidin LL-37, a Defensive Peptide Against Rotavirus Infection

International Journal of Peptide Research and Therapeutics - LL-37 is a 37 amino acid long cationic peptide belonging to the cathelicidin family of antimicrobial peptides. Limited investigations...     

Transmembrane pores formed by human antimicrobial peptide LL-37

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23–33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.     

Expression and purification of a recombinant LL-37 from Escherichia coli

Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic alpha-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as (15)N, (13)C and/or (2)H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C(18) column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the (15)N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform (15)N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies.     

Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin–SUMO dual fusion system

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli.     

Expression and purification of a recombinant LL-37 from Escherichia coli

Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic α-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as ¹⁵N, ¹³C and/or ²H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C₁₈ column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the ¹⁵N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform ¹⁵N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies.     

Targeted expression,purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli

Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro.     

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8⁺ T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8⁺ or CD4⁺ T cells or stimulated CD4⁺ T cells. Of interest, the CD8⁺ cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57BL/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzymes A and B. Granzymes A and B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector, activity of CTL through the secretion of LL-37.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.