title: Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen.
abstract: The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, George Washington University Medical Center, Washington, DC, USA.
authors.forename: Gaddam Narsa
authors.initials: GN
authors.lastname: Goud
authors.forename: Vehid
authors.initials: V
authors.lastname: Deumic
authors.forename: Richi
authors.initials: R
authors.lastname: Gupta
authors.forename: Jill
authors.initials: J
authors.lastname: Brelsford
authors.forename: Bin
authors.initials: B
authors.lastname: Zhan
authors.forename: Portia
authors.initials: P
authors.lastname: Gillespie
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Plieskatt
authors.forename: Eric I
authors.initials: EI
authors.lastname: Tsao
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Bottazzi
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017221:Necator americanus / Q000201:enzymology*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000947:Antigens, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase