Cloning and expression of TNF related apoptosis inducing ligand in Nicotiana tabacum

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand [TRAIL] as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV [Cauliflower Mosaic Virus] helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay [Methylthiazol Tetrazolium Assay]. The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable

Challenges to design and develop of DNA aptamers for protein targets. I. optimization of asymmetric PCR for generation of a single stranded DNA library

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers with high affinity for protein targets and develop an efficient asymmetric PCR amplification. Thus, the influence of factors including annealing temperature, number of amplification cycles, primer ratio, Mg[2+] concentration and the presence of a PCR enhancer on the amplification of the desired product were evaluated. Results obtained by agarose gel electrophoresis showed that the annealing temperature of 64 [degree sign]C, Mg[2+] concentration of 0.25 mM, reverse to forward primer ratio of 15:1, amplification cycle of 25 and the presence L-ectoin as a PCR enhancer with the concentration of 0.4 M were the optimal conditions. Our results supported that the yield of this type of ssDNA production is sufficient for combinatorial screening of aptamers

Overexpression of recombinant human beta interferon [rhINF-beta] in periplasmic space of Escherichia coli

Human Interferon beta [INF-beta] 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-beta-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-beta] 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-beta-D-thiogalactopyranoside [IPTG] was used as a chemical inducer for rhINF-beta production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-beta 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 OD[600nm] = 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-beta into the periplasmic space of E. coli. The obtained yield of the produced rhINF-beta 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

Detection of genetically modi?ed maize in processed foods Sold commercially in Iran by qualitative PCR

Detection of genetically modified organisms [GMOs] in food is an important issue for all the subjects involved in food control and customer's right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt-11 and MON810 events were detected in some maize samples, while, in none of them Bt-176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses

Evaluation of antimicrobial activity of Oudemansiella sp [Basidiomycetes]

Antimicrobial activity of different culture extract of Oudemansiella sp grown on liquid medium [MYGP] were tested. Different fungi [C. albicans, C. lipolytica, Saccharomyces cervisiea, Cladosporium herbarum, and Aspergillus niger] and bacteria [Microccocus luteus, E. coli, Staphylococcus aureus, and Staphylococcus epidermidis] were used as test organisms. Various antimicrobial assay methods including paper disc agar diffusion and microdilution method, were employed to determine possible activity of the extracts. Relative MIC values showed strong activity of the ethyl acetate extract of the fungus against tested bacteria and fungi, especially filamentous fungi