title: Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli.
abstract: CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.
authors.affiliation: Synergen, Inc., 1885 33rd Street, Boulder, Colorado 80301, USA.
authors.forename: G N
authors.initials: GN
authors.lastname: Cox
authors.forename: D
authors.initials: D
authors.lastname: Pratt
authors.forename: D
authors.initials: D
authors.lastname: Smith
authors.forename: M J
authors.initials: MJ
authors.lastname: McDermott
authors.forename: R W
authors.initials: RW
authors.lastname: Vanderslice
mesh_terms: D000069594:Abatacept; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018122:B7-1 Antigen / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D060908:CTLA-4 Antigen; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D018796:Immunoconjugates; D066298:In Vitro Techniques; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D017931:DNA Primers; D018796:Immunoconjugates; D011993:Recombinant Fusion Proteins; D000069594:Abatacept