Lipid Production Capacity of a Newly Characterized Cyanobacterial Strain Synechocystissp. MH01: A Comparative Performance Evaluation of Cyanobacterial Lipid-Based Biodiesel.

BACKGROUND: Cyanobacteria have been the focus of extensive researches because of their high potential for the development of new generations of useful natural compounds with vast applications. For the entire last ten years, a lot of attention has been dedicated to the cyanobacterial lipids as a main source of valuable materials for clean energy production. OBJECTIVES: As there is a direct relationship between biofuel properties and compositional characteristics of fatty acids, a selected lipid-producing cyanobacterial strain was examined and analyzed in terms of fatty acid composition. The biodiesel quality parameters were carefully examined as well. MATERIALS AND METHODS: A cyanobacterial strain was isolated from waterfalls in the northern part of Iran and identified as Synechocystis sp. MH01. The fatty acids profile of the selected strain, as tested in various culture conditions, was analyzed by gas chromatography (GC) and compared with control subjects to further validating the biodiesel quality parameters. RESULTS: The autotrophic cultivation of Synechocystissp. MH01 resulted in biomass and lipid productivity of 109 mg.L-1 day-1 and 22.89 mg.L-1 day-1, respectively. The mixotrophic cultivation of MH01 strain in sucrose-containing medium led to an approximately 1.8 and 1.22 fold increase in biomass and lipid productivity compared with the autotrophic condition. The addition of glycine to BG11 medium caused up to ~1.3 and ~1.18 fold increase in biomass and lipid productivity compared with control subjects. The analysis of qualitative parameters of the biodiesel, as derived from the lipids, indicated that Synechocystis sp. MH01 has a high ability for lipid production under optimal culture conditions. CONCLUSIONS: It seems feasible to evolve the Synechocystissp. MH01 further particularly for more lipid production as a promising primary raw material for biofuel production through fine-tuning of medium composition.

Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli.

BACKGROUND: C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-tumor activities. Recombinant ß-subunit of C-PC (C-PC/ß) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis. OBJECTIVES: Since C-PC/ß has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platensis cpcB gene in a bacterial expression system. This is a significant step for the production of this compound. MATERIALS AND METHODS: The cpcB gene was amplified using specific primers and cloned in a bacterial expression vector, namely pET43.1a+. Gene expression of cpcB was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the dot blotting technique. RESULTS: The SDS-PAGE analysis and dot blotting confirmed the production of recombinant C-PC/ß in the bacterial expression system. Over-expression of cpcB gene was optimized in induction by 1 mM Isopropyl-ß-D-Thiogalactoside (IPTG), after four hours of inoculation at 30°C. CONCLUSIONS: Over-expression of the synthetic CPC/ß protein in the bacterial system (Escherichia coli BL-21) showed that E. coli can be used as a basis for further research to produce this desired protein in large quantities.

Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection.

Background. The aim of this study was to evaluate hepatitis E virus (HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine. Methods. A truncated form of HEV ORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN-γ ELISPOT, and cell proliferative responses following stimulation with the truncated ORF2 protein were assessed in the both groups. Results. The truncated ORF2 protein was able to induce IFN-γ ELISPOT and cell proliferation responses and to produce significant amounts of IFN-γ and IL-12 cytokines, but low amounts of IL-10 and IL-4 cytokines in vitro. These responses were significantly higher in the recovered group compared to the control group. These results indicate the antigenic nature of the truncated ORF2 protein and production of T helper type 1 cytokines. Conclusion. The truncated ORF2 protein can effectively induce significant cellular immune responsesand can be introduced as a potential vaccine candidate. However, further studies are required to evaluate this protein in vivo.

Optimization of Fermentation Conditions for Recombinant Human Interferon Beta Production by Escherichia coli Using the Response Surface Methodology.

BACKGROUND: The periplasmic overexpression of recombinant human interferon beta (rhIFN-ß)-1b using a synthetic gene in Escherichia coli BL21 (DE3) was optimized in shake flasks using Response Surface Methodology (RSM) based on the Box-Behnken Design (BBD). OBJECTIVES: This study aimed to predict and develop the optimal fermentation conditions for periplasmic expression of rhIFN-ß-1b in shake flasks whilst keeping the acetate excretion as the lowest amount and exploit the best results condition for rhIFN-ß in a bench top bioreactor. MATERIALS AND METHODS: The process variables studied were the concentration of glucose as carbon source, cell density prior the induction (OD 600 nm) and induction temperature. Ultimately, a three-factor three-level BBD was employed during the optimization process. The rhIFN-ß production and the acetate excretion served as the evaluated responses. RESULTS: The proposed optimum fermentation condition consisted of 7.81 g L(-1) glucose, OD 600 nm prior induction 1.66 and induction temperature of 30.27°C. The model prediction of 0.267 g L(-1) of rhIFN-ß and 0.961 g L(-1) of acetate at the optimum conditions was verified experimentally as 0.255 g L(-1) and 0.981 g L(-1) of acetate. This agreement between the predicted and observed values confirmed the precision of the applied method to predict the optimum conditions. CONCLUSIONS: It can be concluded that the RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

BACKGROUND: Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. OBJECTIVES: The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. MATERIALS AND METHODS: The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. CONCLUSIONS: In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

Overexpression of Recombinant Human Beta Interferon (rhINF-ß) in Periplasmic Space of Escherichia coli.

Human Interferon ß (INF-ß) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-ß-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-ß) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-ß-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-ß production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-ß protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-ß into the periplasmic space of E. coli. The obtained yield of the produced rhINF-ß was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins.

Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.).

BACKGROUND: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. OBJECTIVES: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. MATERIALS AND METHODS: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. RESULTS: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. CONCLUSIONS: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants.

Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

BACKGROUND: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. METHODS: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. RESULTS: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. CONCLUSIONS: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

Cloning, Transformation and Expression of Human Interferon α2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi).

BACKGROUND: Molecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments. OBJECTIVES: This study reports the transformation of INF α2b gene in tobacco plant for the first time in Iran. MATERIALS AND METHODS: Interferon α2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINFα. pCAMINFα was transferred to E. coli strain DH5α and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon α2b gene was confirmed by dot blotting. CONCLUSIONS: Since no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran.