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

CPN spol. s r.o., Dolní Dobrouc 401, Czech Republic.
Applied Microbiology and Biotechnology (Impact Factor: 3.34). 09/2010; 88(1):167-75. 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.

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    • "To explore the possibility of enhancing efficiency of recombinant human growth hormone production we have modified the E. coli based expression system constructed for the expression of antimicrobial peptide LL-37 [38]. We have fused the codon optimized sequence of hGH with N-terminal thioredoxin gene, which is known to enhance solubility of proteins overexpressed in E. coli cytoplasm [42]. "
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    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 gram per liter 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.
    Protein Expression and Purification 05/2014; 100. DOI:10.1016/j.pep.2014.05.003 · 1.70 Impact Factor
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    • "Another alternative is to control production under an inducible promoter. [27] The heterologously expressed linear AMPs moricin [29], histatin-5 [30], sarcotoxin IA [31], cathelicidin (LL-37) [32] [33] [34], and also AMPs with more complex structural requirements, including disulfide bonds, such as lactoferricin [35], cecropin [36], and β-defensins [37] [38] have all been shown to be functionally active. Table 1 shows an overview of several currently described expression systems of EAP in bacteria. "

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