title: Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris.
abstract: Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Spagnoli
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Bolchi
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Cavazzini
authors.affiliation: German Cancer Research Center, Heidelberg, Germany.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Pouyanfard
authors.affiliation: German Cancer Research Center, Heidelberg, Germany. Electronic address: martin.mueller@dkfz.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Müller
authors.affiliation: Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy. Electronic address: s.ottonello@unipr.it.
authors.forename: Simone
authors.initials: S
authors.lastname: Ottonello
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins
keywords: Human papillomavirus minor capsid protein L2; Pichia pastoris; Pyrococcus furiosus thioredoxin; Recombinant peptide antigens; Secretory production; Vaccine