Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol.
Protein Expr Purif
; 74(2): 211-6, 2010 Dec.
Article
in En
| MEDLINE
| ID: mdl-20600942
ABSTRACT
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 (â¼1 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacteroides fragilis
/
Biochemistry
/
Glutamate-Ammonia Ligase
Language:
En
Journal:
Protein Expr Purif
Journal subject:
BIOLOGIA MOLECULAR
Year:
2010
Document type:
Article
Affiliation country:
South Africa