Targeting to compartments of the endomembranesystem for the accumulation of HIV-1 p24 intobacco plants

Molecular farming aims at producing high-value proteins in plants for pharmaceutical or other industrial use. Tobacco is one of the most used systems in this field of research, offering developed technology for gene transfer and protein expression. Foreign protein stability is a major issue in molecular farming and is strongly dependent on the subcellular compartment of accumulation. We are screening various compartments of the plant endomembrane system for the high accumulation of the HIV-1 p24 nucleocapsid protein, a potential vaccine against the human immunodeficiency virus. Previous work has shown that the localisation of fusions of GFP (Green Fluorescent Protein) to the full length or truncated versions of the transmembrane domain (TMD) of human LAMP1 (Luminal Associated Membrane Protein 1) in tobacco, was detected in the lumen of different compartments of the plant secretory pathway (endoplasmic reticulum -ER-, Golgi Apparatus or plasma membrane), depending on the length of the TMD. In this work, different p24 fusion proteins were designed to accumulate in different compartments of the secretory pathway in tobacco cells. Therefore, the HIV-1 p24 was fused at the N- or C-terminus of the Red Fluorescent Protein (RFP) followed by the different TMDs. Moreover, p24 was also N- or C-terminally fused to the N-terminal domain of maize prolamin γ-zein (zein-p24 and p24-zein). Zein proteins are originally accumulated in ER-derived protein bodies in seeds and previous studies showed the potential of accumulating heterologous proteins in this compartment protecting them from proteases and enhancing their stability. Finally, p24 was fused to the C-terminal tail-anchor of cytochrome b5 (p24-TA), which is expected to be anchored in the ER membrane facing the cytosol. Localisation studies in tobacco protoplasts showed that the constructs containing RFP at the C-terminus of the p24 (p24RFP-TMD) are targeted to the expected compartments (ER, Golgi Apparatus or plasma membrane). However, when the RFP is placed at the N-terminus of p24 (RFPp24-TMD) the fluorescence appears in the tonoplast and the vacuolar lumen, indicating vacuolar delivery and cleavage from the membrane anchor. Transgenic tobacco plants expressing the p24RFP-TMD fusion proteins with the correct targeting to the ER, Golgi and plasma membrane, and also expressing zein-p24, p24-zein and p24-TA were produced. The highest accumulation levels (1% of total soluble protein, TSP) were achieved for p24 containing zein in either N-terminal or Cterminal position. Fusion proteins targeted to the ER showed different accumulation levels if the protein was exposed on the luminal side (p24RFP-TMD, 0.3% TSP) or in the cytosolic side (p24-TA, 0.15% TSP). p24RFP-TMD fusion proteins accumulating in the Golgi apparatus and the plasma membrane showed accumulation levels around 0.15% TSP. The zein fusions formed polymers that were in part difficult to denature even in the presence of SDS, a feature that suggests protein body formation. Pulse-chase experiments indicated that the difference in accumulation of the constructs was mainly due to difference in protein stability. However, RNA blot analysis showed that the zein fusions also lead to increased RNA accumulation. In all cases, the p24 could be released from the fusion tags by digestion with thrombin as the p24 fusion proteins were designed to have a thrombin cleavage site for purification purposes. On the whole, these results highlight the promising approach of targeting HIV-1 p24 to the ER by fusing to the zein domain and provide new information on the relationship between subcellular localisation and stability of integral membrane proteins

Aprendizaje basado en proyectos para motivar al alumnado, trabajar la fragmentación de los saberes en Biología y Geología y visibilizar la mujer en la ciencia en 3º de ESO

El principal objetivo de la educación es el desarrollo integral de las personas, es decir, formar individuos autónomos, con espíritu crítico y emprendedor, competentes, que sepan convivir y que puedan desenvolverse en la sociedad afrontando situaciones cotidianas. En una sociedad cambiante, tecnológica y globalizada, es importante revisar los modelos de enseñanza actuales basados en la clase magistral y la memorización para adaptarlos al presente. Para ello, destacan las metodologías activas que incluyen el Aprendizaje Basado en Proyectos (ABP) y que surgen como respuesta a esta necesidad. El ABP es una metodología didáctica que está centrada en el/la alumno/a, que pasa a ser el/la protagonista de su aprendizaje. Se parte de una situación o problema del mundo real al que el alumnado debe dar respuesta a partir de la investigación y la experimentación, fomentando el aprendizaje significativo. Aunque el aprendizaje por proyectos se ha ido extendiendo en muchos centros educativos de España, supone un reto para el profesorado aprender a trabajar con él para mejorar su labor docente. Este trabajo es una propuesta de ABP para alumnos/as de 3º curso de Enseñanza Secundaria Obligatoria (ESO) con el objetivo de centrar el aprendizaje en el propio alumnado y ofrecer una mayor coherencia en los saberes de Biología y Geología, mejorar la motivación del alumnado despertando vocaciones científicas - sobre todo en las niñas-, y ayudar a visibilizar a la mujer en la ciencia En el proyecto “Doctora, ¿qué me pasa?” se trabajan diferentes bloques de saberes del currículum: la célula, el cuerpo humano, hábitos saludables, salud y enfermedad, el proyecto científico, y ecología y sostenibilidad, integrándolos de forma lógica mediante la consecución de diferentes actividades y prácticas de laboratorio. Es una situación de aprendizaje que se inicia con el reto de determinar qué enfermedad padece un paciente mediante grupos de trabajo cooperativos para posteriormente profundizar en la misma y en todas las estructuras corporales a las que afecta. Es un viaje al centro del cuerpo humano, conectando con la realidad del alumnado y la sociedad de la que forma parte.2022-2

Vaccine Platform for Prevention of Tuberculosis and Mother-to-Child Transmission of Human Immunodeficiency Virus Type 1 through Breastfeeding▿

Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4+- and CD8+-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinant BCG vaccine alone protected against aerosol challenge with M. tuberculosis. Thus, inserting an HIV-1-derived immunogen into the scheduled BCG vaccine delivered at or soon after birth may prime HIV-1-specific responses, which can be boosted by natural exposure to HIV-1 in the breast milk and/or by a heterologous vaccine such as recombinant modified vaccinia virus Ankara delivering the same immunogen, and decrease mother-to-child transmission of HIV-1 during breastfeeding

Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants