Publications by authors named "Somayeh Enayati"

9 Publications

  • Page 1 of 1

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Pathog Dis 2021 Jan;79(1)

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.
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http://dx.doi.org/10.1093/femspd/ftaa075DOI Listing
January 2021

Absence of AfuXpot, the yeast Los1 homologue, limits Aspergillus fumigatus growth under amino acid deprived condition.

World J Microbiol Biotechnol 2020 Jan 30;36(2):28. Epub 2020 Jan 30.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

In Saccharomyces cerevisiae, los1 encodes a nuclear tRNA exporter. Despite the non-essentiality, the deletion of los1 has been shown to extend replicative life span in yeast. Here, we characterized AfuXpot, the los1 homologue in human pathogen Aspergillus fumigatus and found that it is continuously expressed during fungal growth. Microscopic examination of an AfuXpot-GFP-expressing transformant confirmed the nuclear localization of the fusion protein. The targeted gene deletion affirmed the non-essential role of AfuXpot in hyphal growth and sporulation. However, the growth of the deletion mutant was affected by amino acid, but not glucose, deprivation. The susceptibility of the deletant strain to protein and DNA/RNA synthesis inhibitors was also altered. Using bioinformatics tools, some transcription factor binding sites were predicted in AfuXpot promoter. Expression analyses of potential AfuXpot-interacting genes showed a marked down-regulation of sfp1 and mtr10 homologues in ΔAfuXpot strain. Our data demonstrates some conserved aspects of AfuXpot as a tRNA exporter in A. fumigatus.
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http://dx.doi.org/10.1007/s11274-020-2805-8DOI Listing
January 2020

Expression Optimization of Anti-CD22 scFv-Apoptin Fusion Protein Using Experimental Design Methodology

Iran Biomed J 2018 01 10;22(1):66-9. Epub 2017 Jul 10.

Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein.

Methods: Four variables (cell optical density at induction, IPTG concentration, induction temperature, and induction time) were tested using experimental design.

Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield.

Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712387PMC
http://dx.doi.org/10.22034/ibj.22.1.66DOI Listing
January 2018

T7-RNA polymerase dependent RNAi system in Aspergillus fumigatus: a proof of concept study.

FEMS Microbiol Lett 2016 Mar 5;363(5):fnw029. Epub 2016 Feb 5.

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Pasteur Ave, 1316943551 Tehran, Iran

An RNAi system based on T7 RNA polymerase (TRNAP) was designed and examined in Aspergillus fumigatus. This system consists of two elements; an inducible T7RNAP expressing cassette and an AMA1-based episomal RNAi plasmid. These constructs were transformed into the A. fumigatus protoplasts and the efficiency of this system was tested in downregulation of alb1 gene. Upon the induction of T7RNAP expression, the recombinant T7RNAP was able to recognize T7 promoters, which were located on the episomal plasmid and in opposite direction. As a result, the bidirectional transcription of alb1 fragment led to the silencing of the target gene. However, our results demonstrated that this silencing system is unstable and may not be applicable in preparation of RNAi libraries.
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http://dx.doi.org/10.1093/femsle/fnw029DOI Listing
March 2016

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris.

Springerplus 2014 30;3:395. Epub 2014 Jul 30.

Medical Biotechnology department, Fungal Biotechnology group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.
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http://dx.doi.org/10.1186/2193-1801-3-395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124111PMC
August 2014

Cloning and Expression of Gumboro VP2 Antigen in Aspergillus niger.

Avicenna J Med Biotechnol 2013 Jan;5(1):35-41

Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran ; Industrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Background: Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger).

Methods: Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG-protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein.

Results: A number of pyrG (+) positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken.

Conclusion: In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572705PMC
January 2013

NCE102 homologue in Aspergillus fumigatus is required for normal sporulation, not hyphal growth or pathogenesis.

FEMS Microbiol Lett 2012 Apr 13;329(2):138-45. Epub 2012 Feb 13.

Fungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

In Saccharomyces cerevisiae, Nce102 encodes a 173 amino acid transmembrane protein, which acts as a key player in eisosome assembly and plasma membrane organization. Here, we describe the characterization of Nce102 homologue in the human pathogen, Aspergillus fumigatus. Our results demonstrated that AfuNce102 is continuously expressed during fungal growth. In addition, microscopic examination of an AfuNce102-GFP-expressing transformant confirmed the localization of the fusion protein to the endoplasmic reticulum with higher density fluorescence at the tip of the mycelium. During conidiogenesis, the protein was localized to the conidiophores and the conidia. Abnormal conidiation of AfuNce102 deletion mutant suggests a potential role for AfuNce102 in sporulation process.
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http://dx.doi.org/10.1111/j.1574-6968.2012.02513.xDOI Listing
April 2012

Annexin C4 in A. fumigatus: a proteomics approach to understand the function.

J Proteomics 2011 Sep 27;74(10):1950-8. Epub 2011 May 27.

Fungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Annexin C4 has been identified as a new member of fungal annexin family. In search of function, we have generated an annexin C4 disruptant strain of human pathogen, Aspergillus fumigatus. Detailed phenotypic analysis confirmed a non essential role of annexin C4 in the growth and sporulation of this pathogen. We applied a comparative proteomics strategy to understand the possible role of this protein in the fungus. The modification of respiratory chain proteins and stress response proteins suggests the occurrence of a mild oxidative stress in anxC4 disruptant strain. This may indicate a possible anti stress function of annexin C4 in A. fumigatus.
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http://dx.doi.org/10.1016/j.jprot.2011.05.018DOI Listing
September 2011

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments.

BMB Rep 2011 Jan;44(1):34-9

Biotechnology Research center, Pasteur Institute of Iran, Tehran, Iran.

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase®. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase®), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.
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http://dx.doi.org/10.5483/BMBRep.2011.44.1.34DOI Listing
January 2011