Functional surface display of laccase in a phenol-inducible bacterial circuit for bioremediation purposes

Background: Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion [AIDA-I] autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds

Methods: The expression of laccase was regulated by a phenol-responsive promoter [a 54 promoter]. The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds

Results: Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface

Conclusion: Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance

[Cloning and expression of the binding subunit of shiga-like toxin type 2 gene and immunization study in an animal model]

Objective: Enterohemorrhagic Escherichia coli [EHEC] which produces shiga-like toxin type 2 [Stx2] is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome [HUS] and renal failure with a high mortality rate. Stx2 is the major virulence factor of EHEC. Neutralization of toxin by specific antibodies is known to be the best way to prevent and cure HUS. In this study, we describe the cloning, expression, purification, and immunization of the Stx2B subunit which is responsible for toxin binding to the target cell surface

Methods: The Stx2B gene was amplified by PCR and subcloned into a pET28a expression vector and transformed into E. coli BL21-DE3. We evaluated recombinant protein expression and rSTX2B was purified by the Ni-NTA column. The purified rSTX2B was administered subcutaneously to BALB/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX2B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune response was verified by an in vitro assay on HeLa cells and an in vivo assay on mice by challenging them with a lethal dose of Stx2

Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx2 toxin was neutralized by immune mice sera. In the challenge assay, 70% of immunized mice survived

Conclusion: Recombinant rSTX2B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines

[Design and expression of recombinant HER-2 antigen as a marker for detection of breast cancer]

Objective: The incidence of breast cancer is approximately one million which makes this cancer one of the most common among women worldwide. Breast cancer comprises 7% of the total death rate caused by cancers. Several strategies that use tumor-associated antigen [TAA] vaccination and early detection of breast cancer are clinically being developed. Breast cancer is caused by increased over expression of certain genes. HER-2 is a tyrosine kinase receptor in the epidermal growth factor family. The role of HER-2 in breast cancer has been extensively studied. HER-2 is found in 25%-30% of breast cancer patients. Herceptin, a human antibody, is used as a therapeutic target for HER-2. The purpose of this study is to produce recombinant protein HER-2 for early detection of breast cancer cells

Methods: We used specific primers to amplify the HER-2 gene. The amplified gene was cloned into pET28a as an expression vector. Cloning was confirmed by restriction analysis and sequencing. Expression was induced using IPTG and the recombinant protein was analyzed by SDS-PAGE

Results: Cloning of the HER-2 gene was confirmed by enzyme digestion and sequencing. The gene was expressed in E.coli BL21 DE3. The pET-28a vector which contained the HER-2 gene showed a high level of expression. The recombinant protein was confirmed by Western blot analysis

Conclusions: A portion of the HER-2 gene was expressed as a recombinant in E.coli. This could be a good diagnostic test for breast cancer

[Cloning and expression of muc-1 gene in prokaryotic host for early detection of breast cancer]

Background and Aim: Breast cancer is the second most common cause of death among women in the world. Normal mammary gland and breast carcinomas are under the control of regulatory factors, activators and inhibitors inside the breast tissue, as well as a number of growth factors, receptors and proteins outside the breast tissue. Levels of tumor-associated antigens can be used as a predictor in the treatment of this disease. Use of antibodies against MUC1 antigen which is over expressed in 90% of breast cancers is a modern method of treatment. MUC1 tumor antigen disturbs the function of E-cadherin as a cell adhesion molecule. The purpose of this study was to produce MUC-1 recombinant protein for early diagnosis of breast cancer

Material and Methods: A part of MUC-1 gene was amplified by PCR. Then it was cloned into plasmid pET28a in order to be expressed in prokaryotic system. Plasmid pET28a was entered into E.coli BL21DE3 using heat shock method. Cloning process by digestive enzymes and sequence determination were confirmed. Bacteria containing recombinant plasmids were induced by using IPTG and the protein expression was investigated by SDS-PAGE gel

Results: The gene was cloned in the plasmid and the method was confirmed by enzyme digestion and sequencing. Gene expression was confirmed by western blotting

Conclusion: A part of human recombinant MUC-1 gene was produced in E.coli bacteria which can be used as a suitable diagnostic candidate for breast cancer

[Antigenicity of recombinant L7/L12 in patients with brucellosis]

Immune response to recombinant L7/L12, in addition to protective role, may show its importance in detection Brucellosis tests. The aim of this study was to examine antigenicity of recombinant L7/L12 from Brucella abortus by Brucellosis human sera. We amplified L7/L12 gene by polymerase chain reaction [PCR] method and sub- cloned to prokaryotic expression vector pET28a. Escherichia coli BL21-DE3-plySs was transformed with pET28a-L7/L12 and gene expression was induced by IPTG. Recombinant L7/L12 was further analyzed by Western Blot. Sera reactivity of five infected individual were further analyzed against the recombinant L7/L12 protein. The sequencing result was confirmed by Sanger method and it was the same as L7/L12 gene. Escherichia coli BL21 [DE3] pLysS was transformed with pET28a-L7/L12 and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The data also indicated that L7/L12 protein from Brucella abortus recognized by patient sera. Our data showed that recombinant L7/L12 protein can be produced by pET28a in Escherichia coli. This protein was recognized by sera in infected human as an antigen. Therefore, recombinant L7/L12 has same epitopes with natural form of this antigen. Recombinant L7/L12 also seems to be a promising antigen for protection and serologic diagnosis of Human brucellosis

[Production of chimeric tir-intimin protein from E. coli O157:H7 in the tobacco [Nicotiana tobbacum] plant and its immunological evaluation in an animal model]

Escherichia coli [E.coli] O157:H7 is one of the most important pathogenic causes of hemorrhagic colitis in humans. Cattle are the main reservoirs of this bacteria and vaccination is a key mechanism for its control. The intimin, translocated intimin receptor [tir], and EspA proteins are virulence factors expressed by the LEE locus of enterohemorrhagic E. coli. EspA protein is a member of the type III secretion system [TTSS] needle complexes that delivers the tir protein into the host cell. Surface arrayed intimin docks the bacterium to the translocated intimin receptor [Tir]. This intimate linkage is the starting point for attachment and effacing lesions. We hypothesize that the chimeric recombinant forms of two of these three effectors, as edible-based immunogens, would reduce colonization of E. coli O157:H7 in the mice model. We constructed a synthetic gene [it] composed of eae [i] and tir [t] attached together by a peptide linker. The synthetic gene [it] was codon optimized based on the tobacco [Nicotiana tobbacum] plant and cloned into plant expression vectors adjacent to CaMV35S promoters for expression in transgenic tobacco plants. The antigen produced in this plant was orally fed to mice. Immunization of the mice model by the transgenic plant that contained the divalent immunogen showed the presence of IgG antibodies against E. coli O157:H7. This method could be an effective tool for protecting against E. coli O157:H7 hemorrhagic colitis

Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei

Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase [Pwo polymerase] that has proofreading activity. In this study, this microorganism was cultured, its DNA was extracted and the pwo gene polymerase was cloned, expressed and purified. The DNA sequence of the cloned gene was verified by sequencing. The pwo polymerase gene consists of 2,328 bps [775 amino acids with about 90 kD molecular weight]. Cloning was done by GATEWAY Cloning System and for purification of recombinant protein; His6x-Tag was added to the C-terminus of the recombinant protein. We could purify Pwo polymerase enzyme by Ni-NTA resin. PCR assay showed that Pwo polymerase activity is comparable to a commercial Pfu polymerase activity

simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis

In plants, secondary metabolites and polysaccharides interfere with genomic isolation procedures and downstream reactions such as restriction enzyme analysis and gene amplification. The removal of such contaminants needs complicated and time-consuming protocols. In this study, a simple, rapid and efficient method for leaf DNA extraction was optimized. This method use small amount of plant material to reduce inhibitory agents [alkaloids, phenolic]. The procedure involves homogenization of the plant leaf in extraction buffer, incubation at 60°C, extraction by chloroform: iso-amyl alcohol and finally DNA precipitation by cold isopropanol. The results showed that the extracted DNA could be used directly for PCR

Regeneration of glyphosate-tolerant Nicotiana tabacum after plastid transformation with a mutated variant of bacterial aroA gene

Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics is a major hurdle for finding efficient and safe candidates. Herbicide tolerance genes might be attractive alternatives. In this study, a variant form of the 5-enoylpyruvyl shikimate-3-phosphate synthase [EPSPS] gene that harbors glycine at position 96 to alanine and alanine 183 to threonine substitutions and confers higher resistance to the broad-spectrum herbicide, glyphosate, was substituted against the spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. Plastid transformation was carried out using the biolistic delivery procedure while delivery parameters such as rupture disk pressure, bombardment distance, etc had been optimized first. A previous study showed that the glyphosate herbicide imposes lethal effects on the structure and integrity of the plastid membrane, even at low concentrations. In order to overcome this problem, a modified procedure for selection of transplastomic cells was used. A long preculture incubation period followed by a gradual increased in glyphosate concentration led to sufficient expression of the transgene. Tolerant calli were thus regenerated through direct selection of transformed plastids in the presence of the glyphosate

Production of IgY in chicken egg yollk against helicobacter pylori urease using UreC recombinant protein

Helicobacter pylori is a spiral, microaerophilic gram negative bacterium, that multiplies and causes infection in human gastric mucosal layer. H.pylori infection, followed by destruction of gastric epithelial tissue, leads to gastric chronic inflammation, which can cause gastric and peptic ulcers. New approaches have focused on using specific treatments, such as immunotherapy, to eradicate this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. This study is aimed at production of specific IgY against urease UreC subunit. In this study, initially for preparing recombinant UreC, after purification of the genomic DNA, ureC gene was amplified by polymerase chain reaction [PCR]. The PCR product was ligated to pET28a. The recombinant protein was expressed followed by transformation of recombinant construct into E.coli BL21DE3. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to hens. IgY recovered from egg yolk, was purified by PEG precipitation at >70% purity. The purified IgY was analyzed by ELISA and SDS-PAGE. SDS-PAGE analysis revealed a good expression and >70% purification of the recombinant protein. ELISA observation demonstrated high immunogenicity of the recombinant protein. With a view to higher potential of IgY-HpUc in recognition of UreC subunit, the results are in favour of the oral administration of the IgY obtained from hens immunized by H.pylori may provide a novel approach to the management of H.pylori infections

Site-directed mutagenesis, expression and biological activity of E. coli 5-enolpyruvylshikimate 3-phosphate synthase gene

Site-directed mutagenesis [SDM] as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase [EPSPS] gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By designing mutagen primers and overlapping extension method, three kinds of altered bacterial EPSPS enzymes with first, second and both mutations were produced. These modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. These altered genes were cloned under the control of chemically inducible T7 promoter and over expressed in E. coli. Biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate

High level expression of recombinant ribosomal protein [L7/L12] from Brucella abortus and its reaction with infected human sera

Brucellosis, caused by Brucella spp., is an important zoonotic disease that causes abortion and infertility in cattle and undulant fever in humans. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens in animal models. Among Brucella immunogenes, antigen based on ribosomal preparation has been widely investigated. In this study, the immunogenic ribosomal protein L7/L12 gene from Brucella abortus, S19, was amplified by PCR and sub-cloned to prokaryotic expression vector pET28a. Escherichia coli BL21 [DE3] pLysS was transformed with pET28a-L7/L12 and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography with Ni-NTA resin. The concentration of purified recombinant protein calculated to 8 mg/L of initial culture. The integrity of product was confirmed by Western-blot analysis using a standard rabbit anti Brucella abortus ribosomal protein L7/L12 antibody. Sera reactivity of five infected individual were further analyzed against the recombinant ribosomal L7/L12 protein. Data indicated that recombinant ribosomal L7/L12 protein from Brucella abortus was recognized by patient sera