title: Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris.
abstract: The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunologic research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant.
authors.affiliation: Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Hermanrud
authors.forename: Carrie L
authors.initials: CL
authors.lastname: Lucas
authors.forename: Megan
authors.initials: M
authors.lastname: Sykes
authors.forename: Christene A
authors.initials: CA
authors.lastname: Huang
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D023201:CD40 Ligand / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice; D010843:Pichia; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand