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.
Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris / SPAGNOLI, Gloria; BOLCHI, Angelo; CAVAZZINI, Davide; Pouyanfard, Somayeh; Müller, Martin; OTTONELLO, Simone. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 129(2017), pp. 150-157. [10.1016/j.pep.2016.04.012]
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