Speaker
Description
The most widely diagnosed disease globally is cancer, and it has been increasing in incidence and prevalence. The Global Cancer Observatory revealed that in 2022, the number of new cases stood at 20 million, and is set to increase by 77.5 % by 2050. The pro-cancer Retinoblastoma binding protein 6 (RBBP6) is a multi-domain protein that contains the Really Interesting New Gene (RING) domain, which varies across species. Previous multiple sequence alignment studies revealed that the RING domain of the mould Aspergillus niger differs from its human homolog due to the substitution of a cysteine for an aspartic acid. In this study, the A. niger RING domain was successfully expressed in BL21 (DE3) E. coli cells and purified by immobilized-metal affinity and anion-exchange chromatography. The purified protein was characterized using circular dichroism, standard 1D-1H NMR, 1D HET-SOFAST, 1D DOSY, thermal denaturation, thermal shift assays, size-exclusion chromatography with multi-angle light scattering, and differential light scattering. The protein was subsequently set up for crystallization trials. The secondary structural elements of the protein were elucidated by CD, which showed that the protein consisted mostly of β-sheets. 1D NMR revealed that the protein was well-folded and well-structured, with no disordered regions. Thermal denaturation showed the protein's thermal stability, with a Tm of 52°C. SEC-MALS and DLS indicated that the protein is monomeric, with a molecular weight of approximately 9.9 kDa. Crystallization trials yielded crystals under well conditions at 25°C in sodium fluoride, Bis-tris propane, and PEG3350. These conditions were optimized and tested on the ID30A/ MASSIF 1 ESRF beamline, but the protein did not diffract. This study provides a foundation for the structural determination of the A. niger RING domain, with the aim of designing and developing novel anticancer therapeutics.
