فهرست مطالب
Iranian Journal of Biotechnology
Volume:12 Issue: 4, Autumn 2014
- تاریخ انتشار: 1393/12/24
- تعداد عناوین: 9
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Pages 1-9BackgroundPhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene.ObjectivesIdentification of a novel pseudo attP site.Materials And MethodsGenomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably transfected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of minicircle DNAs and followed by sequencing.ResultsA new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site.ConclusionsThis finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications.Keywords: PhiC31 intagrase, Pseudo attP site, Protein expression, Recombination
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Pages 10-16BackgroundOuter inflammatory protein A (OipA) is one of the important adhesins of H. pylori and a valuable candidate for vaccine development. Its gene is under “on-off” switch status which correlates with OipA protein expression.ObjectivesWe aimed to obtain a recombinant OipA clone (with “on” status) from an Iranian clinical isolate.Materials And MethodsAclinical H. pylori-isolate demonstrating high expression for an outer membrane protein (OMP) with an apparent MW of 33-35 kDa was selected. oipA specific primer was designed according to oipA sequences from B8 strain. The purified PCR-product was sequenced and submitted to Gene Bank. The pET-28a plasmid and E. coli DH5α were used for cloning and transformation. The recombinant plasmid was transferred to E. coli BL21 (DE3). Extracted proteins were purified and presence of OipA was confirmed by western blotting using both anti His-tag monoclonal antibody and anti-OipA specific antibody.ResultsThe sequence of the oipA gene and the MW of the purified recombinant OipA protein consisted on 924 bp and 33-35 kDa, respectively. Its identity with other published oipA genes was 92-96%; highest identity was observed with that of a Mexican oipA clone, obtained from a H. pylori strain associated with severe symptoms.ConclusionsRecombinant oipA clone obtained in this work, may be a functional oipA gene with “on” status, associated with more severe outcomes of H. pylori infection.Keywords: Cloning, Helicobacter pylori, Iran, OipA
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Pages 17-25BackgroundEnterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum at various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process.ObjectivesIn this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP.Materials And MethodsAn artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter forexpression of fusion protein EntPMxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distilled water and checked for anti-bacterial activity.ResultsThe fusion protein was highly expressed in insoluble form in E. coli at 37ºC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble contaminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced selfdigestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from soluble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities.ConclusionsThis study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.Keywords: Antimicrobial activity, E. coli expression, Enterocin P, Mxe GyrA mini, intein chitin binding domain (CBD), Purification
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Pages 26-34BackgroundThe refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives.ObjectivesThe purpose of this study was optimization of recombinant human interferon β purification in order to achieve higher efficiency, yield, and a product with a better and more suitable biological activity.Materials And MethodsTriton X-100 and sodium deoxycholate were used to wash the recombinant human interferon-β inclusion bodies prior to solubilization. The inclusion bodies solubilization process was performed by denaturants and reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol.ResultsThe best recovery was obtained in the presence of 0.5% TritonX-100 (v/v). Low concentrations of urea only gave a marginal improvement on the refolding of recombinant human interferon-β. Successful refolding was achieved by gradient elution (decreasing the guanidine-hydrochloride concentration) in the presence of L arginine. Partial purification was also achieved continuously, and recombinant human interferon-β was recovered with 93.5% purity. The interferon prepared in this project was biologically active and inhibited the replication of vesicular stomatitis virus in Hela cells, when compared to the standard interferon.ConclusionsIn this research, the best recovery of inclusion bodies was found at a concentration 0.5 M of Triton X-100, the maximum efficiency of solubility was found in pH 10.5 and the maximum efficiency of refolding was achieved by final buffer containing 2M urea and 0.6 M L-Arg.Keywords: Beta interferon, Inclusion bodies, L, arginine
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Pages 35-40Backgroundα-Amylases (EC 3.2.1.1) are covering approximately 25% of total enzyme market and are frequently used in food, pharmaceutical and detergent industries.ObjectivesThe first ever detailed characterization of amylase from any halophilic Engyodontium album is presented.Materials And MethodsAn extracellular α-amylase was studied from halophilic E. album TISTR 3645. The enzyme was extracted and purified by column chromatography. SDS-PAGE was performed to find the molecular weight of the enzyme. The effects of pH, temperature and salinity on the isolated enzyme were determined. The effects of various additives on enzyme were studied.ResultsThe molecular weight of the amylase was 50 kDa. The enzyme specific activity was 132.17 U.mg-1 with Vmax and Km values of 15.36 U.mg-1 and 6.28 mg.ml-1, respectively. The optimum enzyme activities were found at pH 9.0, 60ºC and 30% (w/v) NaCl. BaCl2, CaCl2, HgCl2 and MgCl2 improved amylase activity. β mercaptoethanol, EDTA, FeCl2 and ZnCl2 decreased enzyme activity.ConclusionsPolyextremophilic characteristics of α-amylase from halophilic E. album TISTR 3645 were revealed during the characterization studies, demonstrating promising features, making it a useful candidate for various industries.Keywords: α amylase, Engyodontium album, Extremozyme, Halophilic fungi
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Pages 41-46BackgroundPectinases are pectin degrading class of enzymes including polygalacturonase (PG), polymethyl galacturonase (PMG), pectate lyase (PEL), and pectin esterase (PE) that are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juices.ObjectivesA highly methylated pectin degrading bacterium from soil covered with fruit waste was isolated and its extracellular pectinase as a novel polymethyl galacturonase was characterized.Materials And MethodsPectin-degrading microorganism screening was performed using agar plates containing pectin as the sole carbon source. The biochemical studies were used to characterize the enzyme.ResultsBacterium with greater PMG activity was a Bacillus sp. based on 16S rDNA sequence analysis and named as a Bacillus sp. strain BR1390. Two steps column chromatography showed a dimeric protein with apparent molecular masses of 104 and 56 kDa, evident by gel filtration and SDS-PAGE. Substrate specificity analysis using various polygalacturonic acid compounds revealed its polymethyl galacturonase (PMG, EC 3.2.1.-) activity. Biochemical studies represent the thermophilic characteristics and reasonable pH stability of the polymethyl galacturonase when using pectin as substrate. The PMG activity significantly enhanced in the presence of most divalent cations such as Ca2+ and Mg2+, but Hg2+ and Fe3+ served as strong inhibitor.ConclusionsOverall, regarding to have suitable activity in acidic conditions and high operational stability of the purified pectinase, the introduced PMG can be an ideal functional substitute for applications in the fruit juice industry, especially in citrus fruits extraction and clarification.Keywords: Dimeric pectinase, Fruit juice industry, Polymethyl galacturonase, Thermostable pectinase
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Pages 47-57BackgroundAeluropus lagopoides is a salt and drought tolerant grass from Poaceae family, distributed widely in arid regions. There is almost no information about the genetics or genome of this close relative of wheat that stands harsh conditions of deserts.ObjectivesThe main aim of this research was to isolation and characterization of salt and drought inducible genes from A. lagopoides by Differential Display Amplified fragment length polymorphism (DD-AFLP) method.Material And MethodsIn this research A. lagopoides was grown under salt or drought conditions and after modifying the DD-AFLP method several fragments were isolated and after nomination their induction was studied by reverse northern blotting.ResultsDD-AFLP led to the improvement of a non-radioactive method for which many parameters were optimized. Having screened approximately 1600 transcript-derived fragments, 1.4 percent of them showed varied expression levels in response to high salt or drought treatments. The relative abundance of twenty one selected differentially expressed fragments was inspected by reverse northern blotting that affirmed the potential of this applied method. Sequence comparisons revealed that some of the isolated genes are involved in osmotic adjustment, regulation of transcription, cation transportation and stress responses. These data clearly show that the modified DD-AFLP method was a successful and reliable approach for the isolation of differentially expressed genes.Keywords: Aeluropus lagopoides, DD, AFLP, Drought, Inducible genes, Salinity
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Pages 58-72BackgroundMost of the amino acids are encoded by more than one codon, termed as synonymous codons. Synonymous codon usage is not random as it is unique to species. In each amino acid family, some synonymous codons are preferred and this is referred to as synonymous codon usage bias (SCUB). Trends associated with evolution of SCUB and factors influencing its diversification in plastomes of genetically distinct Oenothera plastomes have not been investigated so far.ObjectivesIn the present study, major forces that shape SCUB in Oenothera plastomes and putative preferred codons in the protein coding genes (PCG) of plastomes were identified.Materials And MethodsTo unravel various features of SCUB across selected Oenothera plastomes, commonly used codon usage indices such as relative synonymous codon usage (RSCU), synonymous codon usage order (SCUO), effective number of codons (ENC) and codon adaptation index (CAI) were calculated. Correspondence analysis (COA) on RSCU was performed to identify various characteristics of SCUB across different PCG in Oenothera plastomes. Spearman’s rank correlation analysis was adopted to correlate nucleotide contents, codon usage indices and major axes of COA to find out critical parameters in shaping SCUB.ResultsMutational bias due to compositional constraints played crucial role in shaping SCUB as T3 and GC3 contents were in strong negative correlation with all axes of COA. Nevertheless, significant negative correlations between axis 1 and 3 with ENC and CAI respectively, in all species, and narrow distribution of GC contents in neutrality plot, indicate the role of natural selection. Hydropathy score of proteins was found to be influencing SCUB in O. glazioviana as it showed strong negative correlation with axis 2.ConclusionWe concluded that mutational pressure coupled with weak selection influenced SCUB in the examined plastomes of Oenothera. In addition, all examined species of Oenothera exist as disjunct populations in different parts of North America and these populations might have experienced genetic drift as random mutations in small populations that have been fixed over a period of time.Keywords: Genetic drift, Mutational pressure, Oenothera, Selection, Synonymous codon usage bias
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Pages 73-76BackgroundMushrooms have been valued as high tasty nutritional and nutraceutical food throughout the world. At present 270 species of mushrooms are known to have various nutritional and therapeutic properties. Polysaccharides are the primary content of mushrooms that are used for their pharmaceutical properties as immunostimulators.ObjectivesHere, Mushrooms were screened for useful polysaccharides.Materials And MethodsFungal fruiting bodies were collected in rainy season, isolated and maintained in malt agar medium, and screened for their endo- and exo-polysaccharides.ResultsThe mushrooms screened in this study are good producers of exopolysaccharides (EPS) and intracellular polysaccharides (IPS). Ganoderma lucidum was the best producer of polysaccharide among all the isolates.DiscussionsMushroom polysaccharides are well known for their antidiabetic, antitumor and hepatoprotective activities, and so are likely to be used in the preparation of novel drugs. It is suggested that a thorough study on the optimization and characterization of these polysaccharides and biomolecules is essential to exploit this mushroom on an industrial scale.Keywords: Biomolecules, Immunostimulators, Medicinal properties, Mushrooms, Nutraceutical, Polysaccharides