Browsing by Subject "Promoter"

Now showing 1 - 4 of 4
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    Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21
    (Frontiers Media, 2021-06-21) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Martínez Vivancos, Adrián; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
    Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
  • Thumbnail Image
    Publication
    Open Access
    Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21
    (2021-06-21) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Martínez Vivancos, Adrián; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
    Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
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    Regulatory regions of SERPINC1 gene: identification of the first mutation associated with antithrombin deficiency
    (Thieme Gruppe, 2012) Morena-Barrio, Maria Eugenia de la; Antón, Ana Isabel; Martínez Martínez, Irene; Padilla, José; Miñano, Antonia; Navarro Fernández, José; Águila, Sonia; López, María Fernanda; Fontcuberta, Jordi; Vicente, Vicente; Corral, Javier; Medicina
    Antithrombin is the main endogenous anticoagulant. Impaired function or deficiency of this molecule significantly increases the risk of thrombosis. We studied the genetic variability of SERPINC1 , the gene encoding antithrombin, to identify mutations affecting regulatory regions with functional effect on its levels. We sequenced 15,375 bp of this gene, including the potential promoter region, in three groups of subjects: five healthy subjects with antithrombin levels in the lowest (75%) and highest (115%) ranges of our population, 14 patients with venous thrombosis and a moderate antithrombin deficiency as the single thrombophilic defect, and two families with type I antithrombin deficiency who had neither mutations affecting exons or flanking regions, nor gross gene deletions. Our study confirmed the low genetic variability of SERPINC1 , particularly in the coding region, and its minor influence in the heterogeneity of antithrombin levels. Interestingly, in one family, we identified a g.2143 C>G transversion, located 170 bp upstream from the translation initiation codon. This mutation affected one of the four regions located in the minimal promoter that have potential regulatory activity according to previous DNase footprinting protection assays. Genotype-phenotype analysis in the affected family and reporter analysis in different hepatic cell lines demonstrated that this mutation significantly impaired, although it did not abolish, the downstream transcription. Therefore, this is the first mutation affecting a regulatory region of the SERPINC1 gene associated with antithrombin deficiency. Our results strongly sustain the inclusion of the promoter region of SERPINC1 in the molecular analysis of patients with antithrombin deficiency.
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    Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFKB STAT and AP1 transcription factor families
    (Murcia : F. Hernández, 2001) Sevilla, L.; Zaldumbide, A.; Pognonec, P.; Boulukos, K.E.
    Transcription factors play an essential role in determining the fate of a cell by affecting the expression of target genes involved in proliferation, in differentiation and in programmed cell death. Under certain conditions, some of these factors are capable of deregulating expression of genes involved in the cell cycle andlor in programmed cell death resulting in uncontrolled proliferation of the cell. The focus of this review is on the transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein. Since 1999, severa1 papers have implicated members of the Ets, R~~/NFKSBT, N and AP-1 families as transcription factors regulating bcl-x expression. A specific emphasis of these different transcription factor families on bcl-x regulation in hematopoietic cells is discussed