ABSTRACT
Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human ß-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis-Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human ß-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.
Subject(s)
Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Escherichia coli/metabolism , Amino Acid Sequence , Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Candida albicans/drug effects , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli K12/drug effects , Gene Expression Regulation, Bacterial , Humans , Inteins , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tromethamine/analogs & derivatives , beta-Defensins/chemistry , beta-Defensins/genetics , beta-Defensins/metabolism , beta-Defensins/pharmacology , CathelicidinsABSTRACT
<p><b>OBJECTIVE</b>To study the inhibitory activity of Aeschynanthus maculatus on alpha-glucosidase.</p><p><b>METHOD</b>The inhibilitory model of in vitro alpha-glucosidase was established. Active extracts of A. maculatus were isolated and identified bymultiple chromatographic methods, and their molecular structures were identifiied by spectral techniques.</p><p><b>RESULT</b>Seven coumpounts were isolated from A. maculatus and isolated as lupeol(1), stigmasterol(2), ursolic acid(3), stigmast-5,22(E)-diene-3beta-ol(4), beta-daucosterol(5), 3-hydroxy-12-taraxasten-28-oic-acid(6) and oleanic acid(7). Compounds 1 (IC50 25.41 mg x L(-1)),3(IC0 4.42 mg L(-1)),4(IC50 11.50 mg x L(-1)),6(IC50 14.17 mg x L(-1)) and 7(IC50 2.88 mg x L(-1)) had higher inhibitory activities than that of acarbose (IC50 1103.01 mg x L(-1)) as the positive control drug.</p><p><b>CONCLUSION</b>Compound 1-7 were isolated from this plant for the first time. Compound 6 was isolated from Gesneriaceae family for the first time. Compound 7 was isolated from Aeschynanthus genus for the first time.</p>
