Recombinant antigens and adjuvant
The recombinant TSA-1-C4 and Tc24-C4 antigens were obtained from the
Departamento de Biotecnología y Bioingeniería from the Centro de
Investigación y Estudios Avanzados (CINVESTAV) of the Instituto
Politécnico Nacional (IPN), México. Each TSA-1-C4 or Tc24-C4 coding
sequence was cloned into a pET41a+ E. coli expression
vector26,30. The resulting plasmid DNA was transformed
into BL21 (DE3) cells induced with isopropyl-beta-D-1-thiogalactoside
(IPTG) for protein expression. Recombinant proteins were purified by ion
exchange (IEX) and size exclusion chromatography
(SEC)30. The integrity and size of each recombinant
protein was analysed by SDS-PAGE electrophoresis (Supplementary
Fig. 1 ). For cytotoxicity assay, the recombinant proteins were
formulated with the adjuvant E6020 in a stable squalene emulsion (SE).
E6020 Toll-like receptor 4 agonist was acquired through Eisai,
Inc34.