Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin.
Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, PR China.
Lin
L
Wan
Lingyu
L
Zeng
Lihong
L
Chen
Qiang
Q
Huang
Shengfu
S
Li
Yanrong
Y
Lu
Youping
Y
Li
Jingqiu
J
Cheng
Xiaofeng
X
Lu
D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000276:immunology*; D060908:CTLA-4 Antigen; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D054353:Perforin