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

Moon, Jin Woo; Henzler-Wildman, Katherine A.; Ramamoorthy, Ayyalusamy

doi:10.1016/j.bbamem.2006.02.003

Cited by 76 publications

(67 citation statements)

References 37 publications

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“…Given their toxic nature, AMPs are generally expressed as a fusion protein that is not toxic or less toxic to the expression host, and then processed in vitro to release the active AMP. Several strategies have been developed with the aim of eliminating the cytotoxicity of the AMP, improving the solubility and level of the hybrid protein, and facilitating purification of the recombinant protein [Ingham et al, 2005;Morassutti et al, 2005;Moon et al, 2006;Jin et al, 2006;Morin et al, 2006;Niu et al, 2008].…”

Section: Functional Expression By Recombinant Technologymentioning

confidence: 99%

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Dawson

Liu

2009

Drug Development Research

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Sheep myeloid antimicrobial peptide of 29 amino acids (SMAP-29) is one of the most potent antimicrobial peptides known, with a broad spectrum of activity against bacteria, fungi, and viruses. It has also been shown to prevent infections in animals. SMAP-29 is an a-helical cathelicidin that acts rapidly to permeabilize membranes of susceptible organisms. However, it is also cytotoxic and hemolytic to mammalian cells. In this review, all published data on inhibition constants and hemolytic activities of SMAP-29 are brought together for the first time, including data for the peptide of 28 residues that lacks the C-terminal glycine and that also has the new C-terminal residue amidated. Both peptides have been called SMAP-29 in the literature. Antimicrobial activity of SMAP-29 variants (including ovispirins) against Grampositive bacteria is also comprehensively tabulated, and anomalies in the nomenclature of the ovispirins are highlighted. The secondary structure, mode of action, and structure-activity relationships of SMAP-29 are outlined, and the properties that indicate its therapeutic potential are identified; these are its broad-spectrum antimicrobial activity with high potency, rapid biocidal action, limited development of bacterial resistance, synthesis by recombinant technology, ability to bind lipopolysaccharide, and demonstrated protection against infection in animal models. The limitations of SMAP-29 are its high cytotoxicity and hemolytic activity, reduced activity under physiological conditions, and limited evidence for synergistic effects in combination with conventional antibiotics. Strategies with the potential to improve the selectivity toward pathogenic microorganisms over mammalian cells have been devised by the authors and are outlined. Drug Dev Res 70: 481-498, 2009.

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Section: Functional Expression By Recombinant Technologymentioning

confidence: 99%

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Dawson

Liu

2009

Drug Development Research

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“…Direct expression of LL-37 in yeast gave a very low level of the peptide (17). Using bacterial expression systems, we obtained 1.7 mg of recombinant LL-37 from one liter of culture (18), whereas Moon et al (19) reported 0.3 mg. Subsequent studies revealed that the LL-37-containing fusion protein was essentially uncleaved by thrombin at a site adjacent to LL-37 (probably due to peptide aggregation), but could be readily digested by formic acid.…”

mentioning

confidence: 93%

Structures of Human Host Defense Cathelicidin LL-37 and Its Smallest Antimicrobial Peptide KR-12 in Lipid Micelles

Wang

2008

Journal of Biological Chemistry

396

534

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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.The growing drug resistance problem of pathogenic bacteria with traditional antibiotics calls for an urgent search for a new generation of antimicrobial agents. Antimicrobial peptides are ancient and potent weapons of the innate immunity of all life forms (1-4). The recently updated Antimicrobial Peptide Data base collects more than 1228 such peptides (5). In mammals, defensins and cathelicidins are the two major families of antimicrobial peptides. While several cathelicidins were found in animals such as sheep, cow, and pig, only one cathelicidin was identified in humans (6). The precursor proteins of the cathelicidin family share a highly conserved N-terminal "cathelin" domain, but have a highly variable C-terminal antimicrobial region. Upon bacterial insult, human cathelicidin LL-37 (named based on the first two amino acids in the sequence followed by the number of residues in the peptide) is released by proteases from its precursor hCAP-18 (i.e. human cationic antimicrobial protein, ϳ18 kDa). The importance of this host defense peptide to human health is now firmly established. Patients lacking this molecule are more susceptible to infections (7). While cathelicidin knock-out mice are more readily infected (8), expression of additional cathelicidins protects the animals from infection (9). LL-37 also ass...

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“…For instance, the chance-occurrence that more male patients were assigned to the saline group might have contributed to the less favorable outcomes in that group. In addition, we realize that our formulation would probably be improved considerably by adding recombinant human LL-37 26,27 and/or other natural antimicrobial substances that contribute to the innate immunity of airway secretions. 12,13 Also, since nosocomial infections and chronic lung disease are surely of multifactorial pathogenesis, it is unlikely that any one new treatment would eliminate those adverse outcomes.…”

Section: Discussionmentioning

confidence: 99%

ETCare: a randomized, controlled, masked trial comparing two solutions for upper airway care in the NICU

et al. 2007

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Objective: Small quantities of normal saline are sometimes instilled into the endotracheal tube of intubated neonates, to assist with the removal of thick secretions and maintain patency of the endotracheal tube. However, saline is detrimental to the innate immune system of the upper airway mucosa, rapidly unfolding and inactivating antimicrobial peptides such as LL-37. We previously reported the preparation and feasibility testing of 'ETCare', a low-sodium, physiologically based solution for airway care, and we now report results of a randomized, masked, controlled, two-centered study testing ETCare vs sterile saline among 60 intubated NICU patients.Study Design: Sixty intubated NICU patients were randomized to having their airway care with ETCare vs saline. Three hypotheses were tested: (1) tolerance -patients will tolerate ETCare for airway care as well as they tolerate saline, (2) nosocomial infections -ETCare will result in fewer tracheal aspirates where organisms grow and fewer cases of nosocomial sepsis, and (3) chronic lung disuse -ETCare will result in fewer patients discharged home on supplemental O 2 .Result: Thirty NICU patients with an endotracheal tube in place were randomized to receive their airway care with ETCare, and 30 to receive their care with saline. Only the pharmacist was aware of the randomization; the two solutions were visually indistinguishable and were dispensed in identical syringes. Tolerance of the solutions was similar. The ETCare recipients had trends toward fewer positive blood cultures (odds ratios (OR), 0.48; 95% confidence interval (CI), 0.13 to 1.68), and fewer discharges home on supplemental O 2 (OR, 0.43; 95% CI, 0.14 to 1.32; P ¼ 0.075). Conclusion:On the basis of this study and our previous 10-patient feasibility trial, we maintain that, for airway care, intubated NICU patients tolerate ETCare as well as saline. Data from this study can be used in estimating the sample sizes needed for a phase III trial. We speculate that such a trial will demonstrate that, compared with saline, ETCare will result in fewer nosocomial infections and less chronic lung disease.

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

Cited by 76 publications

References 37 publications

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Structures of Human Host Defense Cathelicidin LL-37 and Its Smallest Antimicrobial Peptide KR-12 in Lipid Micelles

ETCare: a randomized, controlled, masked trial comparing two solutions for upper airway care in the NICU

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