2AXON Neuroscience R&D Services SE, Bratislava, Slovakia
Crystallization process requires supersaturated conditions in the macromolecule, which should not significantly perturb its natural state [1]. To solve structure using X-ray crystallography, it is necessary to use high quality crystals. As a first step the initial hits are obtained using different screening conditions in pre-assembled collections. On the market, more than 15 000 conditions from at least 13 vendors are available, therefore it is challenging to find suitable condition to achieve crystal growth [2].
Disordered regions in proteins suppress the regular ordering of protein molecules required for crystal nucleation and growth. Therefore, intrinsically disordered proteins (IDPs) are reluctant to the crystallization; however, it has been shown that complexes of IDPs with binding partners like antibody Fabs are crystallisable [3-4]. IDP tau is principal to neurodegeneration in Alzheimer’s disease. For our work we chose the anti-tau antibody DC25 with epitope on the Lys347-Lys353, which lies in the fourth microtubule-binding repeat of the tau protein [5].
Aim of this work was preparation of crystals suitable for X-ray crystallography. We used recombinant DC25 Fab and truncated form of tau dGAE (contains residues 297-391). For initial crystallization we used Structure screen 1+2 HT-96 (Molecular Dimensions) and JCSG+ screen (Molecular Dimensions). We obtained initial hits for DC25 Fab alone and its complex with tau dGAE, which were further optimised with varying concentration of protein and precipitant. We successfully prepared single crystals larger than 50x50x50 µm3 in different conditions for the DC25Fab as well as for its complex with tau dGAE. We found one condition where both DC25Fab and its complex with tau dGAE crystals grew, which would facilitate interpretation of structural data.