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.     

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for ¹⁵N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.     

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 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.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

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

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.     

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.     

On-resin cleavage of bacterially expressed fusion proteins for purification of active recombinant peptides SK-29, KR-20, LL-29, and LL-23 from human sweat or skin

Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-step chromatographic purification protocol of general use. First, resin-bound fusion proteins were directly subject to efficient upstream thrombin cleavage to release peptide-containing fragments. The resin, resin-bound carrier, and residual uncut fusion proteins were subsequently removed by centrifugation. Second, the peptide-containing fragments were digested with formic acid to release the required peptides followed by reverse-phase HPLC purification. We obtained 1.7 mg of recombinant SK-29, 0.7 mg KR-20, 2.1mg LL-29, and 5.4 mg LL-23, each from one liter of rich medium culture. Analytical HPLC, MS, and NMR indicated high quality of all the purified peptides. Antibacterial assays revealed the minimum inhibitory concentrations (MIC) for SK-29, KR-20, LL-29, and LL-23 are 80, 60, 40, and >150 microM, respectively. The poorest toxicity of LL-23 to Escherichia coli K12 correlates with its higher level of bacterial expression, reduced aggregation tendency, and loss of binding to a yet-to-be-characterized molecular target. Thus, on-resin protein cleavage facilitates the evaluation of peptide aggregation ability and may allow the identification of potential new bacterial targets of antimicrobial peptides. On-resin cleavage may be applied to the release of membrane proteins expressed as fusions.     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.     

Enhanced resistance to bacterial pathogen in transgenic tomato plants expressing cathelicidin antimicrobial peptide

A human cathelicidin antimicrobial peptide (hCAP18/LL-37) is the only member of the mammalian cathelicidin family of proteins that is present in humans. The LL-37 gene was fused to the secretory signal peptide sequence (sp) and a new construct, pSP1-spLL-37, was transformed into tomato. Integration of single copy of the LL-37 was confirmed by TaqMan-PCR and its expression was confirmed by RT-PCR and ELISA assay. The transgenic tomato plants exhibited significant resistance to bacterial soft rot and bacterial spot where it showed strong concerted expression of PR-protein, LTP and AFP1 genes. In vitro screening of protein extracts isolated from the leaves of transgenic plants delimited the growth of P. carotovorum ssp. carotovorum to 15%, and that of X. campestris pv. vesicatoria to 35%. The subcellular localization of LL-37::GFP fusion protein was mainly localized in the cell membrane and cytoplasm. LL-37 expressing-tomato could therefore provide reliable bio-protection against bacterial soft rot and bacterial spot.     

Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni²⁺-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K₁₂D₃₁, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.     

Expression and Purification of Functional Epitope of Pigment Epithelium-Derived Factor in <Emphasis Type="Italic">E. coli</Emphasis> with Inhibiting Effect on Endothelial Cells

PEDF34, a functional epitope of pigment epithelium-derived factor (PEDF), obtained by chemical synthesis previously, shows potential anti-angiogenesis activity described before. We perform a novel method in this study for the expression and purification of recombinant PEDF34 in E. coli, and make it convenient, soluble and high yield to obtain this small peptide of PEDF. Human PEDF34 gene was cloned into the fusion-protein expression vector pGEX-4T-1, and the recombinant plasmid was transformed into E. coli strain BL21-DE3. GST-PEDF34 fusion protein was expressed, purified using chromatograph and identified by Western blotting. The purified fusion protein was digested by thrombin, and the small PEDF34 peptide was isolated by ultrafiltration. Circular dichroism (CD) analysis identified that secondary structure of PEDF34 mainly characterizes as α-helix. The 34-AA small peptide could cell-type-specifically inhibit viability of HUVECs in a dose-dependent manner and induce apoptosis of HUVECs. These results suggested that this type of recombinant PEDF34 may have potential in the treatment of angiogenesis-related diseases such as solid tumor.     

NMR studies of aurein 1.2 analogs

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA > aurein 1.2 >> the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by ¹H NMR spectroscopy, including structural refinement by natural abundance ¹³C^α, ¹³C^β, and ¹⁵N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 Å below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.     

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。     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.     

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.     

Pathogenic Nontuberculous Mycobacteria Resist and Inactivate Cathelicidin: Implication of a Novel Role for Polar Mycobacterial Lipids

Nontuberculous mycobacteria (NTM) are a large group of environmental organisms with worldwide distribution, but only a relatively few are known to be pathogenic. Chronic, debilitating lung disease is the most common manifestation of NTM infection, which is often refractory to treatment. The incidence and prevalence of NTM lung disease are increasing in the United States and in many parts of the world. Hence, a more complete understanding of NTM pathogenesis will provide the foundation to develop innovative approaches to treat this recalcitrant disease. Herein, we demonstrate that several species of NTM show broad resistance to the antimicrobial peptide, cathelicidin (LL-37). Resistance to LL-37 was not significantly different between M. avium that contain serovar-specific glycopeptidolipid (GPL, M. avium^ssGPL) and M. avium that do not (M. avium^ΔssGPL). Similarly, M. abscessus containing non-specific GPL (M. abscessus^nsGPL(+)) or lacking nsGPL (M. abscessus^nsGPL(-)) remained equally resistant to LL-37. These findings would support the notion that GPL are not the components responsible for NTM resistance to LL-37. Unexpectedly, the growth of M. abscessus^nsGPL(-) increased with LL-37 or scrambled LL-37 peptide in a dose-dependent fashion. We also discovered that LL-37 exposed to NTM had reduced antimicrobial activity, and initial work indicates that this is likely due to inactivation of LL-37 by lipid component(s) of the NTM cell envelope. We conclude that pathogenic NTM resist and inactivate LL-37. The mechanism by which NTM circumvent the antimicrobial activity of LL-37 remains to be determined.     

Expression of a novel dual-functional protein--the antimicrobial peptide LL-37 fused with human acidic fibroblast growth factor in Escherichia coli

Human acidic fibroblast growth factor (haFGF) stimulates repair of delayed healing which still remains a tremendously world-wide issue. However, most of the patients with delayed healings have to face another creeping problem - microbial infection, which is one of the most frequent complications that still lead to wound healing failure. LL-37/hCAP-18 is the only cathelicidin-derived antimicrobial peptide found in human with a wide range of antimicrobial activities. In the present study, a novel hybrid protein combining LL-37 with haFGF was designed. The DNA sequence encoding recombination fusion protein LL-37-haFGF was subcloned into the pET-21b vector for protein expression in Escherichia coli strain BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and CM-Sepharose chromatography at a purity of 95.43% as detected by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antimicrobial activity assays showed that the purified LL-37-haFGF had improved antimicrobial activities in vitro compared with LL-37. Methylthiazoletetrazolium (MTT) assay showed that the purified LL-37-haFGF also had a distinct mitogenic activity in NIH 3T3 cells. These data suggests the recombinant protein LL-37-haFGF has pharmaceutical potential for applications in wound healing.