title: Generation of a fusion protein of the extracellular domain of BR3 with the Fc fragment of human IgG1 (sBR3-Fc) in Pichia pastoris as an antagonist for BLyS.
abstract: Elevated levels of B cell-activating factor of the TNF family (BlyS) have been implicated in the pathogenesis of autoimmune diseases in human. Removal of pathogenic B lymphocytes by decoy receptors has demonstrated clinical benefit in both oncological and immunological diseases. In this report, we have constructed vectors for the convenient and rapid expression of the extracellular domain of BR3(sBR3) fused to the Fc fragment (hinge, CH2, CH3) of human IgG1 in the methylotrophic yeast, Pichia pastoris. SDS-PAGE assays of culture broth from a methanol-induced expression strain demonstrated that the recombinant sBR3-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The recombinant protein was purified to >95% using protein A affinity chromatography and size exclusion chromatography steps. Bioactivity of the recombinant sBR3-Fc was confirmed by the ability of the protein to inhibit mouse B lymphocyte proliferation induced by BLyS in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional sBR3-Fc fusion protein for both research and industrial purposes.
authors.affiliation: Laboratory of Molecular Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, Jiangsu, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Cao
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Fang
authors.forename: Houcai
authors.initials: H
authors.lastname: Huang
authors.forename: Ping
authors.initials: P
authors.lastname: Bai
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Zhang
authors.forename: Changliang
authors.initials: C
authors.lastname: Luo
mesh_terms: D000818:Animals; D053265:B-Cell Activation Factor Receptor / Q000235:genetics*; D001402:B-Lymphocytes / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics*; D007155:Immunologic Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
chemical_list: D053265:B-Cell Activation Factor Receptor; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007155:Immunologic Factors; D011993:Recombinant Fusion Proteins