Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli

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 express ed by activated T cells, Both proteins are ligands for the costimulatory mo lecules CD80 and CD86 expressed by activated B cells, macrophages, and dend ritic cells. A fusion protein comprising the CTLA-4 extracellular domain jo ined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune r esponses in vitro and in vivo. Attempts at producing the CTLA-4 extracellul ar domain as an unfused protein have met with limited success. Here we desc ribe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia colt:. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coil and required denaturation, r eduction, and refolding steps to become soluble and assume its proper confo rmation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were t he 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 pote nt than sCTLA-4 monomers at inhibiting T cell activation using a CD80-depen dent in vitro bioassay. (C) 1999 Academic Press.