Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) fusion protein, a novel inummosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4Ig under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4Ig expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4Ig into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4Ig(P)) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4Ig(M)). Recombinant hCTLA4Ig(P) has molecular weight of similar to 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4Ig(M) probably due to the difference of carbohydrate chain structures. Purified hCTLA4Ig(P) was biologically active and was confirmed to suppress T-cell proliferation. (c) 2006 Elsevier Inc. All rights reserved.