title: Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris.
abstract: BACKGROUND
  CD40, also called Bp50, is a novel member of the TNF receptor superfamily. Based on its important role in multiple physiological and pathological processes, the CD40 signaling pathway has become a vital target for treating transplantation, autoimmune diseases and cancers. This study generated a protein fragment that disrupts this signaling pathway.
  
  
  RESULTS
  A DNA fragment encoding the extracellular domain of CD40 (CD40-N) has been codon-optimized and cloned into pPIC9K to create a Pichia pastoris expression and secretion strain. SDS-PAGE and Western blotting assays using the culture media from methanol-induced expression strains showed that recombinant CD40-N, a 27 kDa glycosylated protein, was secreted into the culture broth. The recombinant protein was purified to more than 90 % using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange. Finally, 120 mg of the protein was obtained at a relatively high purity from 3 l supernatant. Binding assay (ITC200 assay) shown the direct interaction of CD40-N and CD40 agonist antibody (G28-5). The bioactivity of recombinant CD40-N was confirmed by its ability to disrupt non-canonical NF-κB signaling activated by CD40 agonist antibody or CD40 ligand and to inhibit ant-CD40 agonist antibody-induced TNF-alpha expression in BJAB cells in vitro. In addition, our data indicate that the protein has curative potential in treating dextran sulfate sodium (DSS)-induced colitis in vivo.
  
  
  CONCLUSIONS
  The results show that the experimental procedure we have developed using P. pastoris can be used to produce large amounts of active CD40-N for research and industrial purposes. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis.
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. zhyu@live.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhan
authors.affiliation: Department of Blood Transfusion, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China. weiyileishuiyan@163.com.
authors.forename: Yilei
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. beimingyu1985@163.com.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Chen
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. hhzhang@sibs.ac.cn.
authors.forename: Haohao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. ddliu@sibs.ac.cn.
authors.forename: Dandan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. fhfdff1@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, Shanghai, China. liur12@fudan.edu.cn.
authors.forename: Rongzeng
authors.initials: R
authors.lastname: Liu
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. chenran@sibs.ac.cn.
authors.forename: Ran
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Blood Transfusion, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China. zhangjun6810@163.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, Shanghai, China. weimo1025@shmu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) and Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Rm. 1126, Biological Research Life Building A, Yueyang Rd 320, Shanghai, 200031, China. xrzhang@sibs.ac.cn.|Collaborative Innovation Center of System Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai, 200240, China. xrzhang@sibs.ac.cn.
authors.forename: Xiaoren
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000818:Animals; D019013:CD40 Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003092:Colitis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010843:Pichia / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D019013:CD40 Antigens; D011994:Recombinant Proteins
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