Pcal n flag

An Escherichia coli (E. coli)‐optimized synthetic gene encoding the E2 ORF from HPV‐16 was cloned into pCAL‐N‐FLAG (Agilent Technologies) containing an N‐terminal calmodulin bind site (CBS). CBS‐E2 and TAT‐CaM were expressed and purified as previously described with slight modifications.²² Briefly, CBS‐E2 was expressed in ArcticExpress (DE3) E. coli cells (Agilent Technologies) and purified by fast protein liquid chromatography using Calmodulin‐Sepharose (GE Healthcare). TAT‐CaM was expressed in BL21(DE3)pLysS E. coli cells (Agilent Technologies) and purified to near‐homogeneity by metal‐affinity chromatography using TALON resin (Takara Bio). After purification, protein constructs were dialyzed into calcium‐containing binding buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, 10% glycerol pH 7.4), sterilized via syringe‐driven filtration through a 0.22 μm filter, flash frozen in liquid nitrogen and stored at −80°C until use. Samples were collected at each stage of the purification process in 2% SDS buffer and subjected to gel electrophoresis as previously described.²⁷ Elutions were further subjected to immunoblot analysis as previously described using an HPV 16 E2 monoclonal primary antibody TVG 621 (ThermoFisher) and goat anti‐mouse HRP conjugated secondary (ThermoFisher).²⁷