title: Cloning, high yield overexpression, purification, and characterization of AlgH, a regulator of alginate biosynthesis in Pseudomonas aeruginosa.
abstract: The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate. Production of alginate by P. aeruginosa is tightly regulated at the transcriptional level. AlgH, a putative transcriptional regulator, is involved in regulating alginate biosynthesis as well as nucleoside diphosphate kinase activity and succinyl coenzyme A synthetase activity in P. aeruginosa. Sequence homologues are found in many bacterial species. Here, we describe a method for high level overexpression and high yield/high purity production of AlgH for biophysical and functional studies. The algH gene was cloned and AlgH was overexpressed in Escherichia coli using a commercially available vector with an inducible T7 promoter. We purified the recombinantly produced protein using a rapid classical purification scheme. The yield of purified protein, either isotopically labeled for NMR studies or unlabeled, is excellent (30-37 mg of purified protein per liter of minimal media culture), as is the purity (>95% pure). Analysis of the secondary structure using circular dichroism and NMR indicates that the protein is comprised of both beta-sheet and alpha-helical secondary structural elements. Heteronuclear NMR spectra indicate that AlgH is a monodisperse, folded globular protein. This rapid, high yield, and high purity method for AlgH production will permit further biophysical characterization of this protein including high resolution structural studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
authors.forename: Ramona J
authors.initials: RJ
authors.lastname: Bieber Urbauer
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: Sara E
authors.initials: SE
authors.lastname: Rosasco
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Hattle
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Cowley
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
mesh_terms: D000464:Alginates; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D013389:Succinate-CoA Ligases; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015854:Up-Regulation
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D014157:Transcription Factors; D020723:Glucuronic Acid; D013389:Succinate-CoA Ligases