Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.     

Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

Reconstitution of Fragmentary Form of Thermostable Alanine Racemase

The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure.     

Removal of Phthalate Esters in Soil Column Inoculated with Microorganisms

The removal of phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) was efficiently effected by inoculating and retaining viable cells of Nocardia erythropolis, a bacterium known capable of rapidly degrading phthalate esters, in soil column. When an influent containing 3000 ppm of DEHP was passed through a column inoculated with Nocardia erythropolis, the eluent from the column was gas-chromatographically free of DEHP after 1 day. Residual DEHP on the support after 32 days in the column inoculated with Nocardia erythropolis was only 0.14% against the total amount of DEHP fed, whereas it was 5.2% in the uninoculated column. Microorganisms capable of utilizing DEHP were isolated from the inoculated and un- inoculated columns after 32 days operation and identified. The DEHP utilizing microorganisms in the inoculated column were found to belong to Nocardia erythropolis, Nocardia restricta and Pseudomonas putida (Biotype B), and those in the uninoculated column to Nocardia erythropolis, Pseudomonas putida (Biotype A and B) and Pseudomonas acidovorans. In particular, strain 1-1 of Nocardia erythropolis isolated from the inoculated column was morphologically and biochemically identical with the inoculated Nocardia erythropolis S-l. Ratio of all Nocardia erythropolis to total cells recovered increased from 10.8% immediately after inoculation to 27.2% after 32 days in inoculated column.     

Overexpression, Purification, and Refolding of a Porphyromonas gingivalis Cysteine Protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.     

Recombinant Bacterial Expression of the Lysozyme from the Tobacco-Hornworm <Emphasis Type="Italic">Manduca sexta</Emphasis> with Activity at Low Temperatures

A gene coding for lysozyme from the insect Manduca sexta (Ms-lyz) was expressed in Escherichia coli. The protein was produced as an insoluble cytoplasmic inclusion body which was denatured in 8 M guanidine-HCl, renatured and purified by affinity and ion-exchange chromatography. The N-terminal sequence and the activity of the recombinant protein against Micrococcus luteus confirmed that correct expression had occurred. When Ms-lyz activity was compared to hen egg white lysozyme, the insect lysozyme was active at lower temperatures. These results demonstrate the feasibility of producing a disulfide-bonded lysozyme enzyme in bacteria and suggest that the insect Ms-lyz is an interesting system for further development of an antibacterial functional at low temperatures.     

The antibacterial effect of a polyhydroxylated fucophlorethol from the marine brown alga,Fucus vesiculosus

An antibacterial compound was isolated from the brown alga Fucus vesiculosus. Purification consisted of extraction of plant material with 0.05% trifluoroacetic acid, concentration on a C₁₈ cartridge, and reverse-phase high performance liquid chromatography on a C₁₈ semi-preparative column. The isolated compound exhibited antibacterial activity against both the Gram-positive and the Gram-negative bacteria tested. Killing studies conducted indicated that the activity was bactericidal. The compound showed no haemolytic effect against human red blood cells. Results obtained by electrospray ionization mass spectrometry indicated that the antibacterial activity was caused by a polyhydroxylated fucophlorethol.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Production and purification of a novel antibiotic peptide,adenoregulin, from a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l^–1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni²⁺-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.     

Antibacterial potential of Forsythia suspensa polysaccharide against resistant Enterobacter cloacae with SHV‐12 extended‐spectrum β‐lactamase (ESBL)

In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 10⁴ Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV‐12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV‐12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 μg/mL) was tested against E cloacae bearing SHV‐12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV‐12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration‐dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV‐12 ESBL via inhibition of SHV‐12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug‐resistant bacterial pathogen.     

Expression, Purification, and Refolding of Active Recombinant Human E-selectin Lectin and EGF Domains in Escherichia coli

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni²⁺ affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe^X (K _d = 321 nM, B_max = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.     

Cycloamylose as an efficient artificial chaperone for protein refolding

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.     

Antibacterial activity of recombinant hCAP18/LL37 protein secreted from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) was expressed in Pichia pastoris and its antibacterial activity was tested against pathogenic bacteria. The full length ORF of hCAP18/LL37 was cloned into the pPICZaA vector followed by integration into the genomic AOX1 gene of P. pastoris. Agar diffusion assay demonstrated that the different hCAP18/LL37 transformants showed various antibacterial activities against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis. The secreted form of hCAP18/LL37 exhibited its maximum activity after 72 h incubation with 2% methanol in MM media, not in BMM. This result suggests that the yeast secreted expression system can be used as a production tool of antimicrobial peptides for industrial or pharmaceutical application.     

Production and purification of a cecropin family antibacterial peptide,hinnavin II,in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly, hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni²⁺-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin II in its biologically active form.     

Antibiotic‐Potentiating Activity of Phanostenine Isolated from Cissampelos sympodialis Eichler

Cissampelos sympodialis Eichler is well studied and investigated for its antiasthmatic properties, but there are no data in the literature describing antibacterial properties of alkaloids isolated from this botanical species. This work reports the isolation and characterization of phanostenine obtained from roots of C. sympodialis and describes for the first time its antimicrobial and antibiotic modulatory properties. Phanostenine was first isolated from Cissampelos sympodialis and its antibacterial activities were determined. Chemical structures of the alkaloid isolate were determined using spectroscopic and chemical analyses. Phanostenine was also tested for its antibacterial activity against standard strains and clinical isolates of Escherichia coli and Staphylococcus aureus. Minimal inhibitory concentration (MIC) was determined in a microdilution assay and for the evaluation of antibiotic resistance‐modifying activity. MIC of the antibiotics was determined in the presence or absence of phanostenine at sub‐inhibitory concentrations. The evaluation of antibacterial activity by microdilution assay showed activity for all strains with better values against S. aureus ATCC 12692 and E. coli 27 (787.69 mm ). The evaluation of aminoglycoside antibiotic resistance‐modifying activity showed reduction in the MIC of the aminoglycosides (amikacin, gentamicin and neomycin) when associated with phanostenine, MIC reduction of antibiotics ranging from 21 % to 80 %. The data demonstrated that phanostenine possesses a relevant ability to modify the antibiotic activity in vitro. We can suggest that phanostenine presents itself as a promising tool as an adjuvant for novel antibiotics formulations against bacterial resistance.     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

Chemical Composition,Antibacterial, Antioxidant and in Vitro Antidiabetic Activities of Essential Oils from Eruca vesicaria

This work describes the study of the chemical composition and bioactivity of the essential oils (EOs) of the different organs (leaves, flowers, stems and roots) from Eruca vesicaria. According to the GC and GC/MS analysis, all the EOs were dominated by erucin (4‐methylthiobutyl isothiocyanate) with a percentage ranging from 17.9 % (leaves) to 98.5 % (roots). The isolated EOs were evaluated for their antioxidant (DPPH, ABTS and β‐carotene/linoleic acid), antibacterial and inhibitory property against α‐amylase and α‐glucosidase. Most EOs exhibited an interesting α‐glucosidase and α‐amylase inhibitory potential. The roots essential oil was found to be the most active with IC₅₀ values of 0.80±0.06 and 0.11±0.01 μg mL^?1, respectively. The essential oil of roots exhibited the highest antioxidant activity (DPPH, PI=92.76±0.01 %; ABTS, PI=78.87±0.19; and β‐carotene, PI=56.1±0.01 %). The isolated oils were also tested for their antibacterial activity against two Gram‐positive and three Gram‐negative bacteria. Moderate results have been noted by comparison with Gentamicin used as positive control.