Preparation and bioactivity evaluation of streptavidin-tagged human interferon- inducible T cell alpha chemoattractant bifunctional fusion protein.
- Author:
Xiaoling XU
1
;
Ying LIU
;
Qingge CHEN
;
Tongliang HUANG
;
Jimin GAO
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Biotinylation; Blotting, Western; Cancer Vaccines; Cell Line, Tumor; Chemokine CXCL11; chemistry; Chromatography, Affinity; Humans; Interferons; chemistry; Mice; Plasmids; Recombinant Fusion Proteins; biosynthesis; chemistry; Streptavidin
- From: Journal of Southern Medical University 2015;35(12):1715-1720
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity.
METHODSpET24a-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.
RESULTSSA-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.
CONCLUSIONSWe successfully obtained SA/hI-TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.
