Expression and purification of bioactive high-purity mouse monokine induced by IFN-gamma in Escherichia coli.
MIG (monokine induced by IFN-gamma) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. The non-tagged recombinant murine MIG (rMuMIG) is therefore required to facilitate the functional studies of this important chemokine. Here we present the use of a bacteria expression system to produce non-tagged rMuMIG. The coding sequence for MIG was cloned into the pET28a (+) vector that was transformed into Escherichia coli BL21 (DE3). Expression of rMuMIG was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and resolved in Urea buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose cation exchange chromatography. The final preparation of the rMuMIG was more than 99% pure as measured by capillary electrophoresis and SDS-PAGE analysis. The biological activity of rMuMIG was demonstrated in a murine spleen cell chemotaxis assay with ED50 30 ng/ml. Further experiments showed that rMuMIG could inhibit proliferation of mouse bone marrow cells in vivo.
Lu
Huili
H
Laboratory of Regenerative Medicine, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China.
Yu
Mei
M
Sun
Ye
Y
Mao
Wenwei
W
Wang
Qun
Q
Wu
Mingyuan
M
Han
Wei
W
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D054370:Chemokine CXCL9; D019743:Chemokines, CXC / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002633:Chemotaxis; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007371:Interferon-gamma / Q000494:pharmacology / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D015846:Monokines / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology
D054370:Chemokine CXCL9; D019743:Chemokines, CXC; C485969:Cxcl9 protein, mouse; D015846:Monokines; D011994:Recombinant Proteins; D007371:Interferon-gamma