Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol.
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.
van Rooyen
J M
JM
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa. jason@science.uct.ac.za
Abratt
V R
VR
Belrhali
H
H
Sewell
B T
BT
D001441:Bacteroides fragilis / Q000201:enzymology*; D001671:Biochemistry / Q000379:methods*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase