Title
Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris
Author
Gloria Spagnoli, Angelo Bolchi, Davide Cavazzini, Somayeh Pouyanfard, Martin Müller, Simone Ottonello
Year
2016
Journal
Protein Expression and Purification
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 inP. pastoris were tested and found to be identical to those of the same antigen produced inEscherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastorisrepresents 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.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Thermal stability, Biochemistry