Human interleukin-12 (hIL-12), a heterodimeric cytokine comprised of p35 and p40 subunits, was generated previously via tobacco cell suspension culture at a maximum concentration of only 175 mu g/L. In order to improve the productivity, the rice a-amylase 3D promoter (Ramy3D) Was utilized in order to express recombinant protein at high levels under sucrose starvation conditions. The hIL-12 gene was cloned into the rice expression vector under the control of the Ramy3D promoter. Transgenic rice calli were prepared via particle bombardment-mediated transformation and were screened for rhIL-12p70 expression using ELISA. Western blot and bioassay results confirmed that rhIL-12p70 was expressed and secreted into the culture medium as a functionally active heterodimeric form, and the productivity of rhIL-12p70 was estimated as 12.7mg/L, which is approximately 73-fold higher than was observed with the tobacco cell culture system [Kwon TH, Sec, JE, Kim J, Lee JH, Jang YS, Yang MS. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol Bioeng 2003;81:870-5]. However, the secreted rhIL-12p70 proved unstable in the medium, and degraded rapidly after day 13. In order to stabilize the secreted rhIL-12p70, three stabilizing polymers were tested (polyethylene glycol, polyvinyl pyrrolidone, and gelatin). Gelatin was the most effective in stabilizing the secreted rhIL-12p70. After the addition of 2% (w/v) gelatin, the maximum rhIL-12p70 concentration reached 31.0 mg/L, representing a 2.5-fold increase over the control. (C) 2009 Elsevier Inc. All rights reserved.