[Preparation and bioactivity evaluation of streptavidin-tagged human interferon- inducible T cell alpha chemoattractant bifunctional fusion protein].

OBJECTIVE

To prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity.

METHODS

pET24a-SA-hI-TAC/pET21a-hI-TAC-SA plasmids were constructed and expressed in BL21. SA-hI-TAC and hI-TAC-SA fusion proteins were purified by Ni-NTA affinity chromatography, refolded by dialysis and identified by Western blotting. The bifunctionality of the fusion proteins (biotin-binding function and hI-TAC activity) was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively.

RESULTS

SA-hI-TAC/hI-TAC-SA fusion proteins were expressed at about 12% and 25% of the total bacterial protein, respectively. The two fusion proteins had a purity of about 85% and 90% after purification, and their purity reached 98% after purification with S-100 gel filtration chromatography. Both of the fusion proteins were efficiently immobilized on the surface of biotinylated mouse bladder cancer MB49 cells (91.3% for SA-hI-TAC and 98.8% for hI-TAC-SA). SA/hI-TAC induced lymphocyte chemotaxis in a dose-dependent manner, and hI-TAC-SA showed a stronger chemotactic effect than SA-hI-TAC.

CONCLUSIONS

We successfully obtained SA/hI-TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.