Fig. 4

Production and purification of GP40 of Cryptosporidium parvum in Toxoplasma gondii. a Purification process of GP40 in T. gondii using Strep-TactinXT. The parasite lysis supernatant was coupled to a Strep-Tactin resin column. Due to the low affinity of Strep-Tactin for non-specific interactions, other proteins were readily washed away even under mild physiological conditions. The purified recTgGP40 was eluted by the introduction of low concentrations of desthiobiotin. This step, based on competitive displacement, enhances specificity while maintaining the overall buffer conditions (such as pH and ionic strength) unaltered. As a result, highly purified recTgGP40 was obtained and the function of the target protein was preserved; 10 mM NaOH solution was used to regenerate the Strep-Tactin column. b The purified recTgGP40 protein was visualized by Coomassie Brilliant Blue staining of SDS-PAGE gels (sodium dodecyl sulfate–polyacrylamide gel electrophoresis): Lane 1: purified recTgGP40 before concentration; lane 2: purified recTgGP40 after concentration. The red arrow indicates the recTgGP40 glycoprotein band. c The purified protein was analyzed by Western blotting with mAb HA. Lane 1: recTgGP40 after purification