Biochemistry and Microbiology

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Browsing Biochemistry and Microbiology by Author "Akapo, Olufunmilayo Olukemi"

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    Recombinant expression, purification and structural characterization of aspergillus niger ring finger domain using nuclear magnetic resonance spectroscopy
    (University of Zululand, 2018) Akapo, Olufunmilayo Olukemi
    e retinoblastoma binding protein 6 (RBBP6) is a 250 kDa nuclear protein which contains an N-terminal DWNN domain and a conserved RING finger domain. Previous studies determined the solution structure of the human RING finger domain using heteronuclear NMR spectroscopy. A bioinformatics analysis using the human RING finger from RBBP6 as searching bait revealed an unchanged zinc ion coordination pattern in all organisms except in three organisms: Saccharomyces cerevisiae, Pichia pastoris and Aspergillus niger. Therefore, structural characterization of the amino acid substitution observed in A. niger RING finger domain will add to our understanding of the biological role played by the RING finger domains. In this study, in-silico characterization of the A. niger RING finger using different bioinformatics tools was carried out. Subsequently, the RING finger was sub-cloned into a pGEX-6P-2 vector and expressed as a GST-RING fusion protein. GST-Agarose affinity chromatography was used to partially purify this protein to homogeneity. Thereafter, biophysical characterization of the purified protein was carried out using Fourier transform Infrared spectroscopy (FTIR) and 1D NMR. The predicted three-dimensional structure of A. niger was done using the SWISS-MODEL server and visualized on Chimera showed a consensus structure containing two α-helices and 3 random coils. This result is consistent with the secondary structure prediction performed using PSIPRED v3 and JPred 4. The predicted 3D model was validated by analyzing a Ramachandran Plot, which indicated 88.0 % of the residues are in the most favoured region. This is very close to the 90% widely accepted mark of 3D models indicative of excellent quality models. Added to this, eight B-cell epitopes were predicted from the protein with four being continuous epitopes, while the remaining four are discontinuous epitopes. Thereafter, A. niger RING finger domain was sub-cloned into a pGEX-6P-2 protein expression vector and heterologously over-expressed as a GST fusion protein, which was later purified using GST-Agarose affinity chromatography. The biophysical analysis of the purified protein using a 1D proton NMR spectroscopy data revealed the appearance of a large chemical shift dispersions range from 1.0 ppm and -1.0 ppm in the methyl group and also 8.5 ppm downfield the amide region, thereby confirming the foldedness of the protein. Taken together, these results showed that the A. niger RING finger domain is very well folded and can be subjected to structural determination using heteronuclear NMR spectroscopy after collecting triple resonance coherence data.
University of Zululand