Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5?mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1?l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195?Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50)?=?16-64?μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.     

The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins

Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.     

Annoquinone-A,an antimicrobial and cytotoxic principle from Annona montana

A new naturally occurring phenanthrene-1,4-quinone, annoquinone-A, along with parietin (physcion) and β-sitostenone were isolated from the stem bark of Annona montana. The structure of annoquinone-A was elucidated by spectral methods and synthesis. Annoquinone-A demonstrated potent antimicrobial activity against Bacillus subtilis and Micrococcus luteus as well as cytotoxicity in the KB (ED₅₀ = 0.16 μg/ml) tissue culture assay. β-Sitostenone also showed significant cytotoxicity.     

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.     

Proteinase inhibition and antioxidant activity of selected forage crops

The hypothesis of the possible therapeutic potential of selected species of forage crops is discussed. Extracts from genotypes of Anthyllis sp., Astragalus sp., Coronilla sp., Lotus sp., Medicago sp., Melilotus sp., Onobrychis sp. and Trifolium sp. were prepared and tested for proteinase inhibition and antioxidant activities. We found that Trifolium pratense accession POLKIE99-3 expressed the highest relative trypsin inhibition activity (80.0%) compared to standards. The highest thrombin inhibition activity (81.4%) was detected in the Medicago sativa old cultivar Hodoninka, whereas the highest relative urokinase inhibition activity (62.5%) was expressed by the local population Nitranka. Relatively high antioxidant properties of Trifolium sp. accessions, Trifolium pratense genetic resources SVKZAH98-40, were of interest. Results of this study confirmed that there are significant differences in proteinase inhibition and antioxidant activity among important selected agricultural crops. The present paper may also be the starting point of the research aimed for development of new functional food and nutraceuticals, and/or of the research focused on new secondary metabolites with potential as bioactive compounds.     

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins

Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, -globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (M_rs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH₂-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with M_rs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the -globulin and one of the new low-molecular-weight proteins of M_r 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH₂-terminal amino acids.Abbreviations HMW high molecular weight - IgE immunoglobulin E - LMW low molecular weight - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - RP-HPLC reversed-phase high-performance liquid chromatography - SDS sodium dodecyl sulphate We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica.     

The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins

A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7.     

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides.The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel.This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.     

Mating,male Ixodes scapularis express several genes including those with sequence similarity to immunoglobulin-binding proteins and metalloproteases

We created a cDNA library from mating, male Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate genes common to feeding female and mating male I. scapularis ticks. A total of seven unique cDNAs were identified in this screen. One cDNA had sequence similarity to the IGBP-MC gene from Rhipicephalus appendiculatus, and another cDNA potentially encodes a protein with similarity to metalloproteases. RT-PCR, using RNA isolated from male and female I. scapularis ticks, confirmed that these genes are expressed in male, but not female ticks. The remaining five cDNAs did not match any sequences in the GenBank database. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.     

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

Apyrase,streptavidin-binding proteins,and antimicrobial activity in Pisum sativum

The 49 kD apyrase (EC 3.6.1.5), streptavidin-binding proteins, and antimicrobial activity in the subcellular fractions from different seed parts of Pisum sativum L. var. Alaska were examined. Except cotyledons, all subcellular fractions contained 49 kD apyrase, and a considerable relationship was found between 49 kD apyrase and NTPase activities that increased with increasing time of germination. The bulk of 49 kD apyrase and NTPase activities was found in the nucleus pellets and cytoskeleton-enriched fraction, indicating their physiological importance. At 72 h of germination, all subcellular fractions of primary stems have a greater amount of 49 kD apyrase and NTPase than primary leaves and much more than primary roots and cotyledonary stalks. All seed parts showed antimicrobial activities, and the bulk of inhibition activities was found in the cytoskeleton-enriched and nucleus pellets, which was greater in the primary stems and leaves than in other parts. Current findings reveal that apyrases have important roles in metabolic activities in all parts of the pea plants except cotyledons. Cotyledons contained much streptavidin-binding proteins, which might have different physiological roles than apyrases.     

Interaction of human complement with Sbi, a staphylococcal immunoglobulin-binding protein: indications of a novel mechanism of complement evasion by Staphylococcus aureus

Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and beta(2)-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption.     

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an α-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo , we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.     

Solution structure of LCI, a novel antimicrobial peptide from Bacillus subtilis

LCI, a 47-residue cationic antimicrobial peptide (AMP) found in Bacillus subtilis, is one of the main effective components that have strong antimicrobial activity against Xanthomonas campestris pv Oryzea and Pseudomonas solanacearum PE1, etc. To provide insight into the activity of the peptide, we used nuclear magnetic resonance spectroscopy to determine the structure of recombinant LCI. The solution structure of LCI has a novel topology, containing a four-strand antiparallel β-sheet as the dominant secondary structure. It is the first structure of the LCI protein family. Different from any known β-structure AMPs, LCI contains no disulfide bridge or circular structure, suggesting that LCI is also a novel β-structure AMP.     

Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.