Co-solute assistance in refolding of recombinant proteins

Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct refolding. Co-solutes play a major role in refolding process and can be classified according to their function as, aggregation suppressors and folding enhancers. This study presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial process.Key words: Refolding, protein aggregation, low-molecular-weight additives, arginine

Expression of human interferon gamma in Brassica napus seeds

Expressions of heterologous proteins in suitable plant tissues and targeting it into subcellular compartments using specific signals have been studied. Seed-based platforms are among those that allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass. In this study, we used seed specific promoter (Napin) and C-terminal KDEL sequence to express human therapeutic protein, interferon gamma (IFN_y) in Brassica napus seeds. Kozak sequence was linked to the 5' end of the IFN_y gene to increase the level of expression. The constructed cassette was transformed into rapeseed. Presence and expression of the transgene were confirmed in the transformants by polymerase chain reaction (PCR) and sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Analysis of transgenic plants by enzyme-linked immunosorbent assay (ELISA), dot blot and western blot indicated that IFN_y protein is being expressed in B. napus seeds and is as active as the standard IFN_y. Our results indicate that plant seeds have tremendous potential for production of recombinant proteins as ‘natural bioreactors’.Key words: Interferon gamma, KDEL retention signal, seed specific promoter, Brassica napus, recombinant proteins

CD8+ T Cells as a Source of IFN-γ Production in Human Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses

Inhibition of Leishmania Major PTR1 Gene Expression by Anti-sense in Escherichia Coli

Background:Protozoa related to Trypanosome family including Leishmania,synthesize enzymes to escape from drug therapy.One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase(DHFR)Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more.Methods: PTR1 gene  was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E.coli strain M15.SDS-PAGE and western blot analysis were carried out to analyze the expression.Results:Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done.SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells.Conclusion:Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

Molecular Cloning and Expression of Iranian Leishmania major Pteridine Reductase 1

Background: Leishmaniasis is an endemic disease in 88 countries. Reports on Leishmania drug resistance are growing in number. The mechanism of unresponsiveness against glucantime in Iranian cutaneous leishmaniasis has not yet been characterized. To begin the first step in finding an anti-Leishmania chemotherapy, we prepared recombinant L. major PTR1 enzyme and characterized its activity by enzymatic assay. Methods: Leishmania promastigote DNA was extracted and the ptr1 gene amplified using specific primers. The PCR product was cloned in pQE30 expression vector, transformed into E.coli and expressed. The recombinant protein was purified, its enzymatic activity was assayed and anti-PTR1 antibody prepared in rabbit. Results: The PCR product of ptr1 gene was sequenced and deposited in GenBank. The amino acid sequence of Iranian L.major PTR1 was compared with other Leishmania PTR1 and showed some identities and diversities. Purified protein was reacted by anti PTR1 antibody in gel diffusion and western blot assy. Enzyme activity of purified recombinant PTR1 was 38 nmol/min per 0.4 mg of protein and it showed pteridine reduction by PTR1 Conclusion: We cloned and expressed Iranian L. major ptr1 gene and assayed its enzymatic activity. This enzyme will be used for further investigation about Leishmania antifolate therapy that is effective against PTR