فهرست مطالب

Iranian Journal of Biotechnology
Volume:10 Issue: 4, Autumn 2012

  • تاریخ انتشار: 1391/07/23
  • تعداد عناوین: 7
|
  • Vida Tafakori, Ibrahim Torktaz, Mohsen Doostmohammadi, Gholamreza Ahmadian Page 231
    Cell-surface display is the expression of peptides and proteins on the surface of living cells by fusing them to functional components of cells which are exposed to the environment of cells. This strategy can be carried out using different surface proteins of cells as anchoring motifs and different proteins from different sources as a passenger protein. It is a promising strategy for developing novel whole cell factories. Surface engineered cells have many potential uses ranging from medical to environmental applications. This review focuses on different strategy and applications of microbial surface display.
    Keywords: Environmental, Medical, Microbial Surface Display
  • Mozhgan Rasti, Zahed Khatooni, Zoherh Mostafavi, Pour Page 240
    p53 is a key tumor suppressor gene that is targeted for inactivation during human tumorigenesis. In this study, we produced and characterized polyclonal anti-human p53 antibody. The cDNA encoding the complete human p53 protein was cloned into pGEX-4T-1 and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST). The rabbits were immunized with the purified p53 recombinant protein. The obtained antisera were purified to increase the specificity of recognition. The sensitivity and specificity of the produced antibody was analyzed by enzyme-linked immunosorbent, immunoblot, immunofluorescence and chromatin immunoprecipitation assays. Enzyme-linked immunosorbent assay showed that immunization with purified GST-p53 produced the high titer (1:10000) polyclonal antibodies with high specificity. Anti-p53 antibody allowed the sensitive detection of native p53 protein in immunoblotting, immunofluorescence and chromatin immunoprecipitation assays. Our results showed that anti-GST-p53 antibody provides a good means for studying the p53 expression pattern and its binding ability to other proteins in tumors.
    Keywords: GST, p53, Tumor, Immunoassays, Anti, p53 antibody
  • Mohammad Askari, Fazel Gorjipour, Zohreh Sharifi, Mohammad Morad Farajollahi Page 249
    So far, recombinant antigens of HIV-1, the etiologic cause of Acquired Immunodeficiency Syndrome (AIDS), have been widely used for the diagnosis and vaccine development. P17 or the matrix protein formed by the proteolytic cleavage of gag is strongly antigenic and is as conserved and immunogenic as p24. In some cases, antibodies to p17 are more prevalent than antibodies to p24 and the decline in the level of p17 antibodies is an earlier prognostic marker for disease progression than decline in the level of antibodies to p24. The aim of this study was to clone and express the gag derived p17 protein in soluble form in E. coli and then assess the immunoreactivity of produced recombinant p17. DNA sequence encoding p17 matrix protein was cloned from PTZ-gag53-IR vector that has the complete gag polyprotein sequence. The T7-promoter-based expression system used in this study was TOPO directional cloning strategy that expressed p17 matrix protein in fusion with thioredoxin in E. Coli. Purification of produced recombinant protein has been done using Ni-NTA nickel chelating agarose beads. Sequencing result of the cloned sequence showed that it belongs to CRF35_AD subtype of HIV-1 which is highly prevalent in Iran and Afghanistan. The immunoreactivity of produced recombinant p17 to sera from infected individuals showed 93.8% sensitivity and 100% specificity.
    Keywords: HIV, 1 CRF35, AD, Cloning, Recombinant protein, p17 antigen, Fusion protein
  • Ali Asghar Karkhane, Bagher Yakhchali, Ferdous Rastgar Jazii, Jafar Hemmat, Parvin Shareati, Mahvash Khodabandeh, Alireza Zomorodipoor Page 255
    Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene (btl2) was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors (pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA) were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies.
    Keywords: Bacillus thermocatenulatus Lipase, capillary isoelectric focusing, Inclusion bodies, Periplasmic space, Tandem mass spectrometry
  • Mohammad Ghomi Avili, Mohammad Hassan Fazaelipoor, Seyed Ali Jafari, Seyed Ahmad Ataei Page 263
    This paper presents a comparison between batch and three different sets of fed batch fermentations for rhamnolipid production by Pseudomonas aeruginosa. The batch run was performed with 500 ml of culture medium having the initial glycerol and sodium nitrate concentrations of 30 and 8.3 g/l, respectively. For a fed batch run with nitrogen source in feed, 250 ml of the nitrogen excluded culture medium was in the bioreactor initially, and 250 ml culture medium containing 16.6 g/l sodium nitrate was fed to the bioreactor continuously. A similar procedure was repeated for fed batch runs with carbon, and phosphorus source in feed. Statistical analysis showed that fed batch runs were better than batch in term of rhamnolipid production, and among the fed batch runs the maximum amount of rhamnolipid. (4.12 g Rhamnose Equivalent/l) was for the fed batch run with the carbon source in feed.
    Keywords: Pseudomonas aeruginosa, Rhamnolipid, Batch process, Fed batch process, Statistical analysis
  • Shahnaz Esmaeli, Abdolamir Allameh, Mohammad Sajad Emami Aleagha, Somaieh Kazemnejad, Masoud Soleimani Page 270
    Currently several studies are being carried out on various properties of mesenchymal stem cells (MSCs) however there are a few investigations about drug metabolizing properties of these cells. The aim of this study was to measure the key factors involved in drug metabolism in human bone marrow MSCs. For this purpose, cellular glutathione (GSH), glutathione S-transferase (GSTs) and cytochrome P450 class 3A4 (CYP3A4) were detected in these cells. Results showed that CYP3A4 and GSTA1-1 mRNA are not detectable in MSCs however mRNA specific for GSTP1-1 was considerably expressed in MSCs. GSH content and GST activity was also detected in MSCs. These data suggest that human bone marrow MSCs possess the drug metabolizing activity which may be useful in handling drugs and chemotherapeutic agents passing to the bone marrow.
    Keywords: Human bone marrow mesenchymal stem cells, Drug metabolism, Glutathione S, transferases, cytochrome P450
  • Elmira Hajinia, Seyed Safa, Ali Fatemi, Ali Asghar Karkhane, Ali Akbar Safekordi, Bagher Yakhchali Page 275
    Human granulocyte macrophage colony stimulating factor (hGM-CSF) has many therapeutic applications. In this study, in order to verify the purification process, the effect of carbon source, IPTG concentration and post-induction time on the secretion of recombinant hGM-CSF into the culture medium by recombinant Escherichia coli during high cell density cultivation were evaluated by using the Taguchi statistical method. The results indicated that glucose, 1mM IPTG and a time of 6 h post-induction, represented optimum conditions. The secreted hGM-CSF, overall volumetric productivity and purified hGM-CSF were 373 mg/l, 18 mg/l/h and 63 mg/l, respectively.
    Keywords: Escherichia coli, Fed, batch, High cell density cultivation, Human granulocyte macrophage colony stimulating factor, Optimization, Purification