title: Cloning and high level expression of the biologically active extracellular domain of Macaca mulatta CD40 in Pichia pastoris.
abstract: The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.
authors.affiliation: Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China.
authors.forename: Shengyun
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wan
authors.affiliation: Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China.
authors.forename: Jingqiu
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Key Lab of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, 610041, China. Electronic address: xiaofenglu@yahoo.com.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019013:CD40 Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D023201:CD40 Ligand / Q000737:chemistry; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000502:physiology; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia; D011485:Protein Binding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D019013:CD40 Antigens; D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D023201:CD40 Ligand
keywords: CD40; Chronic inflammatory disease; Immunosuppression; Pichia pastoris; Rhesus monkey