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 (similar to 1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GM enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coil 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. (C) 2010 Elsevier Inc. All rights reserved.