Generation and Characterization of Recombinant HTP-3 Proteins

Recombinant HTP-3 protein was generated by cloning full-length cDNAs of htp-3 into pTrcHis Topo (Invitrogen, catalog no. K4410-01) bacterial expression vectors to generate N-terminally 6× His-tagged proteins (Das et al. 2020 (link)). Mutant HTP-3^S285A protein was generated through site-directed mutagenesis as described (Arur et al. 2009 (link)). Positive clones were then sequence verified. Recombinant proteins were expressed in BL21(DE3) (Sigma-Aldrich) cells at 37°C by using 1-mM isopropyl β-d-1-thiogalactopyranoside (dioxane free) for 3 h. Proteins were then purified by using Ni–nitrilotriacetic acid resin (Thermo scientific, no. 88221). The expression of proteins was confirmed by Western blot analysis with anti-His (Sigma-Aldrich, no. H1029). In vitro kinase assay was performed using purified ERK2 kinase (New England Biolabs, catalog no. P6080S), and the purified recombinant proteins according to the methods previously described (Arur et al. 2009 (link)). After phosphotransfer, the proteins were resolved onto a 10% SDS–polyacrylamide gel electrophoresis (Bio-Rad, catalog no. 4561033). The gel was then dried at 60°C under vacuum for 1 h and exposed to the autoradiographic film (Sigma-Aldrich, catalog no. 864 6770) for 4 h at −80°C followed by the development of the film using the Kodak X-OMAT 2000A processor machine.