Browsing by Author "Belrhali, H"

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    Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol
    (Elsevier, 2010) van Rooyen, J M; Abratt, V R; Belrhali, H; Sewell, B T
    Glutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large (1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.