title: Expression and purification of recombinant human Mig in Escherichia coli and its comparison with murine Mig.
abstract: Monokine induced by IFN-γ (Mig) is a member of CXC-chemokines and recruits T-lymphocytes to activate the immune response. In recent years, it has raised much interest in the areas of autoimmune disease and allograft rejection, as the production of recombinant human Mig (rHuMig) would be of considerable significance for both research and potential clinical use. Here we report the expression, preparation and characterization of non-tagged recombinant human Mig (rHuMig) using a prokaryotic expression system. Following expression in Escherichia coli (E. coli) BL21, the 103 amino acid residue of rHuMig was purified from bacteria inclusion bodies with a one-step S-Sepharose cation exchange chromatography. The product was immunologically characterized via Western blot and its purity was determined via SDS-PAGE and silver staining to be above 99%, with an endotoxin level <0.5EU/μg via a chemotaxis assay, rHuMig demonstrated chemotactic activity on mouse spleen lymphocytes with an ED50 of 15 ng/mL. Additionally, using a proliferation assay, rHuMig significantly inhibited proliferation of the human bladder cell line T24. In vivo experiments revealed that rHuMig could inhibit mouse bone marrow mononuclear cells cycling into the S-phase and reduced intestinal cell proliferation. Our results demonstrate that rHuMig is fully functional in the mouse model.
authors.affiliation: Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China.
authors.forename: Lan
authors.initials: L
authors.lastname: Qian
authors.forename: Shunying
authors.initials: S
authors.lastname: Zhu
authors.forename: Jiaqing
authors.initials: J
authors.lastname: Shen
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Han
authors.forename: Jin
authors.initials: J
authors.lastname: Gao
authors.forename: Mingyuan
authors.initials: M
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yu
authors.forename: Huili
authors.initials: H
authors.lastname: Lu
authors.forename: Wei
authors.initials: W
authors.lastname: Han
mesh_terms: D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation; D054370:Chemokine CXCL9 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D002634:Chemotaxis, Leukocyte; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000166:cytology
chemical_list: D054370:Chemokine CXCL9; D011994:Recombinant Proteins