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Available from: Kamran Shehzad Bajwa
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ABSTRACT: The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional gene promoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.
Available from: Nadia Hassan
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ABSTRACT: Human interferon alpha-2b (hIFNα2b) is the most important member of interferon family. Escherichia coli, yeast, mammalian cell cultures and baculovirus infected insect cells have been used for expressing recombinant human interferon. Recently Pichia pastoris based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered, an important aspect to get the optimum expression of recombinant protein which may vary from one protein to another. In present study, we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b expressing cassettes were created via in vitro multimerization approach. P. pastoris host strain X-33 was transformed with these expression cassettes. Groups of P. pastoris clones transformed with different copies of IFNα2b expression cassette were screened for intrachromosomal integration. IFNα2b expression level of stable transformants was checked. Copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that increase in copy number generally have positive effect on expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. This was also observed that increase in drug resistance of a clone not guarantee its high expression, as integration of marker gene do not always correlate with integration of gene of interest. This article is protected by copyright. All rights reserved.
Available from: Zareen Fatima
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