title: Process development for production and purification of the Schistosoma mansoni Sm14 antigen.
abstract: The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 °C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250  mg L-1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.
authors.affiliation: Cornell University-Ludwig Institute for Cancer Research Partnership Laboratory, Ithaca, NY, United States.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Damasceno
authors.affiliation: Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY, United States.
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
authors.affiliation: Cornell University-Ludwig Institute for Cancer Research Partnership Laboratory, Ithaca, NY, United States. Electronic address: cab10@cornell.edu.
authors.forename: Carl A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D050612:Fatty Acid Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015870:Gene Expression; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012550:Schistosoma mansoni / Q000235:genetics* / Q000276:immunology; D014612:Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000947:Antigens, Helminth; D050612:Fatty Acid Transport Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins; D014612:Vaccines; C068709:SM14 protein, Schistosoma mansoni