Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures.
Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive 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 hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.
Lee
Song-Jae
SJ
Boryung Pharmaceutical Co. Ltd., Ansan 425-120, Republic of Korea.
Park
Cheon-Ik
CI
Park
Mi-Young
MY
Jung
Hahn-Sun
HS
Ryu
Wuk-Sang
WS
Lim
Sang-Min
SM
Tan
Hyun-Kwang
HK
Kwon
Tae-Ho
TH
Yang
Moon-Sik
MS
Kim
Dong-Il
DI
D000069594:Abatacept; D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D006801:Humans; D018796:Immunoconjugates / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000378:metabolism*; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D012275:Oryza / Q000166:cytology / Q000378:metabolism; D030821:Plants, Genetically Modified; D000516:alpha-Amylases / Q000235:genetics
D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D000069594:Abatacept; D000516:alpha-Amylases