title: Improved expression and purification of the Helicobacter pylori adhesin BabA through the incorporation of a hexa-lysine tag.
abstract: Helicobacter pylori is a pathogenic bacterium that has the remarkable ability to withstand the harsh conditions of the stomach for decades. This is achieved through unique evolutionary adaptations, which include binding Lewis(b) antigens found on the gastric epithelium using the outer membrane protein BabA. We show here the yield of a recombinant form of BabA, comprising its putative extracellular binding domain, can be significantly increased through the addition of a hexa-lysine tag to the C-terminus of the protein. BabA was expressed in the periplasmic space of Escherichia coli and purified using immobilised metal ion affinity and size exclusion chromatography - yielding approximately 1.8 mg of protein per litre of culture. The hexa-lysine tag does not inhibit the binding activity of BabA as the recombinant protein was found to possess affinity towards HSA-Lewis(b) glycoconjugates.
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, Nottinghamshire NG7 2RD, United Kingdom.
authors.forename: Naim
authors.initials: N
authors.lastname: Hage
authors.affiliation: AstraZeneca R&D, Alderley Park, Cheshire SK10 4TG, United Kingdom.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Renshaw
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, Nottinghamshire NG7 2RD, United Kingdom.
authors.forename: G Sebastiaan
authors.initials: GS
authors.lastname: Winkler
authors.affiliation: AstraZeneca R&D, Macclesfield, Cheshire SK10 2NA, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Gellert
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, Nottinghamshire NG7 2RD, United Kingdom.
authors.forename: Snow
authors.initials: S
authors.lastname: Stolnik
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, Nottinghamshire NG7 2RD, United Kingdom. Electronic address: franco.falcone@nottingham.ac.uk.
authors.forename: Franco H
authors.initials: FH
authors.lastname: Falcone
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016480:Helicobacter pylori; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012394:Rosaniline Dyes / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; C400429:BabA protein, Helicobacter pylori; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue; D008239:Lysine
keywords: BabA; Helicobacter pylori; Hexa-lysine tag; Lewis(b); Outer membrane porin; Periplasmic expression; Polycationic tag