Browsing by Subject "Recombinant proteins"

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    Compound heterozygosity involving Antithrombin Cambridge II (p.Ala416Ser) in antithrombin deficiency.
    (Elsevier, 2013-03-10) Águila Martínez, Sonia; Martínez-Martínez, Irene; Collado, Miriam; Llamas, Pilar; Antón, Ana I.; Martínez-Redondo, Consuelo; Padilla, José; Miñano, Antonia; Morena Barrio, María Eugenia de la; García-Avello, Ángel; Vicente García, Vicente; Corral de la Calle, Javier; Medicina Interna
    Background: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. Objectives: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. Methods: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. Results and Conclusions: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity
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    Development of biological tools to assess the role of TMPRSS4 and identification of novel tumor types with high expression of this prometastatic protein
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Villalba, Maria; Lopez, Lissett; Redrado, Miriam; Ruiz, Tamara; de Aberasturi, Arrate L.; de la Roja, Nuria; Garcia, David; Exposito, Francisco; de Andrea, Carlos; Álvarez Fernández, Emilio; Montuenga, Luis; Rueda, Paloma; Rodriguez, Maria Jose; Calvo, Alfonso
    Metastatic spread is responsible for the majority of cancer deaths and identification of metastasisrelated therapeutic targets is compulsory. TMPRSS4 is a pro-metastatic druggable transmembrane type II serine protease whose expression has been associated with the development of several cancer types and poor prognosis. To study the role and expression of this protease in cancer, we have developed molecular tools (active recombinant proteins and a polyclonal antibody) that can be used for diagnostic purposes and for testing anti-TMPRSS4 drugs. In addition, we have evaluated TMPRSS4 protein expression in several cancer tissue microarrays (TMAs). Full length and truncated TMPRSS4 recombinant proteins maintained the catalytic activity in two different expression systems (baculovirus and E. coli). Sensitivity of the rabbit polyclonal antisera against TMPRSS4 (ING-pAb) outperformed the antibody most commonly used in clinical settings. Analysis by immunohistochemistry in the different TMAs identified a subset of adenocarcinomas, squamous carcinomas, large cell carcinomas and carcinoids of the lung, in which TMPRSS4 expression may define aggressive tumors. In conclusion, our biological tools will help the characterization of TMPRSS4 activity and protein expression, as well as the evaluation of anti-TMPRSS4 drugs. Future studies should determine the clinical value of assessing TMPRSS4 levels in different types of lung cancer.
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    Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.
    (Springer Nature, 2019-09-04) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
    Background: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overfow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to fnd the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two diferent approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. Results: We evaluated protein overexpression in E. coli BL21 wt and fve mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efcient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fvefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. Conclusion: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fuxes to diferent pathways and afect the fnal yield of the recombinant protein bioprocess. Thereby, we obtained a fvefold increase in protein production and an efcient use of the resources employing the most suitable strain and culture conditions.
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    Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations
    (2014-04-15) Águila Martínez, Sonia; Navarro Fernández, José Luis; Bohdan, N.; Gutiérrez Gallego, R.; Morena Barrio, María Eugenia de la; Vicente García, Vicente; Corral de la Calle, Javier; Martínez-Martínez, I.; Medicina Interna; Facultad de Medicina