Summary of key activities carried out by the Programme

Throughout last year we have continued working on the expression and purification of BRCA1. We have carried out several trials of expression in transformed insect cells in collaboration with the group of Dr. Montoya, which have prompted us to build a recombinant bacmid to increase the purification yield.

Regarding cloning and expression of PINS complexes (MUD:PINS:GαI and INSC:PINS:GαI), we have cloned several proteins into an expression vector for P. pastoris system (in collaboration with the group of Dr. González). We are currently checking protein expression in eukaryotic system to try to overcome the insolubility problems detected in prokaryotic systems.

At the level of structural characterization by electron microscopy and image processing of centrosomal components, we have analyzed XMAP215 protein and its interaction with coiled-coils domains of Maskin in collaboration with the group of Dr. Montoya. The preliminary 2D models obtained have allowed us to elucidate some structural features such as internal domains as well as the oligomerization state of different XMAP215.

We have also analyzed by electron microscopy several tubulin cofactors such as TBCE, and several complexes such as TBCB:TBCE, TBCB:TBCE:αTubulin and TBCD:Arl2, in collaboration with the group of Dr. Zabala. We are currently improving the 2D and 3D information generated so far.

We have trapped single centrosomes using optical tweezers and have characterized their average size and electrical charge. We have contributed, by electron microscopy, to the finding that the phosphorylation of -tubulin induced by the protein SADB regulates centrosome duplication.

In the field of electron tomography of centrosomes, and in collaboration with the group of Dr. Carazo, we have worked on the purification of thymus centrosomes from sheep and goat with the aim of generating a 3D reconstruction of this structure.

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