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SUMMARY:Recombinant Production and Biophysical Characterization Towards St
 ructural Determination of Aspergillus niger RING finger domain
DTSTART;VALUE=DATE-TIME:20260608T173000Z
DTEND;VALUE=DATE-TIME:20260608T180000Z
DTSTAMP;VALUE=DATE-TIME:20260606T092152Z
UID:indico-contribution-10354@events.saip.org.za
DESCRIPTION:Speakers: Mary George (University of Johannesburg)\nThe most w
 idely 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 increas
 e by 77.5 % by 2050. The pro-cancer Retinoblastoma binding protein 6 (RBBP
 6) is a multi-domain protein that contains the Really Interesting New Gene
  (RING) domain\, which varies across species. Previous multiple sequence a
 lignment studies revealed that the RING domain of the mould Aspergillus ni
 ger differs from its human homolog due to the substitution of a cysteine f
 or an aspartic acid. In this study\, the A. niger RING domain was successf
 ully expressed in BL21 (DE3) E. coli cells and purified by immobilized-met
 al affinity and anion-exchange chromatography. The purified protein was ch
 aracterized using circular dichroism\, standard 1D-1H NMR\, 1D HET-SOFAST\
 , 1D DOSY\, thermal denaturation\, thermal shift assays\, size-exclusion c
 hromatography with multi-angle light scattering\, and differential light s
 cattering. 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 reveal
 ed that the protein was well-folded and well-structured\, with no disorder
 ed regions. Thermal denaturation showed the protein's thermal stability\, 
 with a Tm of 52°C. SEC-MALS and DLS indicated that the protein is monomer
 ic\, with a molecular weight of approximately 9.9 kDa. Crystallization tri
 als yielded crystals under well conditions at 25°C in sodium fluoride\, B
 is-tris propane\, and PEG3350. These conditions were optimized and tested 
 on the ID30A/ MASSIF 1 ESRF beamline\, but the protein did not diffract. T
 his study provides a foundation for the structural determination of the A.
  niger RING domain\, with the aim of designing and developing novel antica
 ncer therapeutics.\n\nhttps://events.saip.org.za/event/274/contributions/1
 0354/
LOCATION:
URL:https://events.saip.org.za/event/274/contributions/10354/
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