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

ArticleinApplied Microbiology and Biotechnology 88(1):167-75 · September 2010with22 Reads
DOI: 10.1007/s00253-010-2736-7 · Source: PubMed
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 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-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.
    • "The values for all Cronobacter strains were below the value of 9.23 μg·ml -1 obtained for E. coli ML-35p. The MIC values for Cronobacter were in the range obtained for the majority of Gram-negative bacteria strains, 0.9–7.7 μg·ml -1 [13, 17]. In next experiments, antimicrobial activity of LL-37 in liquid media was studied using the same strains. "
    [Show abstract] [Hide abstract] ABSTRACT: Cronobacter spp. is responsible for rare but fatal cases of infections in neonates and immunocompromised infants. The reconstituded powdered infant formula (PIF) is one of main sources of newborn infections. The aim of our study was to assess application of recombinant human cathelicidin LL-37, an antimicrobial peptide, alone and in combination with bacteriophages against Cronobacter in PIF. The minimal inhibitory concentration (MIC) of LL-37 determined by radial diffusion method for ten representative Cronobacter strains ranged from 2.56 μg·ml-1 to 8.83 μg·ml-1, and the values were below MIC of E. coli standard strain. By testing the antimicrobial activity in liquid growth medium, substantial inhibition of strains was observed at 30 μg·ml-1 LL-37, whereas 20 μg·ml-1 showed only moderate effect. However, higher peptide concentrations were necessary to inhibit Cronobacter in reconstituted PIF. By using 50 μg·ml-1 and 100 μg·ml-1 LL-37, numbers of cells decreased after 4-hour cultivation by approx. 70% and 96%, respectively. Finally, simultaneous application of LL-37 and bacteriophage Dev-CS-701 resulted in synergistic effect, as a 1 300-fold reduction of cell numbers in PIF was observed. Application of the two antimicrobial agents thus facilitated their reduced dosing and decreased the probability of generation of phage-resistant cells.
    Full-text · Article · Jan 2015
    • "Glycoprotein 120 (gp120) is part of the HIV envelope protein and its fragment (target analyte in current study) has been used for the induction of anti-HIV neutralization antibodies [24, 25] . Although there are published literatures reporting the use of RP-HPLC for the purification of LL-37 [26, 27], to the best of our knowledge, there is no validated RP-HPLC method for determining the concentration of LL-37 or gp120 fragment alone or simultaneously . The current study is the first to apply a validated RP- HPLC method for the simultaneous quantification of LL-37 and gp120 fragment in polymeric gel dosage forms. "
    [Show abstract] [Hide abstract] ABSTRACT: Recently, a growing number of macromolecules such as peptides and proteins have been formulated into various microbicide formulations for the prevention of sexually transmitted infections. However, a fast and reliable high-throughput method for quantitating peptide/protein in polymer-based microbicide formulations is still lacking. As a result, we developed and validated a reversed-phase high-performance liquid chromatography method for the quantitation of gp120 fragment and LL-37 simultaneously in various microbicide gel formulations. This method was capable of detecting a limit of linearity (regression coefficient of 0.999) for gp120 fragment and LL-37 within a range of 0.625–80 and 1.25–80 µg mL−1, respectively. The lower limit of quantification for gp120 fragment and LL-37 was 1.14 and 0.31 µg mL−1, respectively. Method validation demonstrated acceptable intra- and inter-day RSD % (<5 %) and accuracy (95.67–100.5 %). Formulating both peptides into polymeric pharmaceutical gel formulations showed high extraction efficiency (in the range of 95.90 ± 3.03 to 111.45 ± 2.51 %). Using this method, we were able to separate and identify the forced degraded products from both peptides simultaneously without affecting the quantitation of both peptides in the polymeric dosage forms. Furthermore, this method was able to detect and separate degradants that were unable to be revealed using gel eletrophoresis.
    Full-text · Article · Oct 2014
    • "Strain Genotype Reference DH5a FÀ u80lacZZ DlacZYA-argF) U196 endA1 recA1 hsdR17 (rkÀ,mk+) supE44 thi-1 gyrA96 relA1 phoA Invitrogen BL21(DE3) F – ompT gal dcm lon hsdS B (r B À m B À ) k(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Novagen RV308ai Su-lacX74 gal::ISII(OP308) araBAD::T7 RNAP strA [38] "
    [Show abstract] [Hide abstract] ABSTRACT: Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation , was confirmed by employing a Nb2 cell line proliferation assay.
    Conference Paper · Jul 2014
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