Expression of Human Fc-fused Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures

초록

Glucocerebrosidase (GCD) is a hydrolytic lysosomal enzyme indicated for enzyme replacement therapy (ERT) of Gaucher disease. Fc-fusion results in increased serum half-life, stability and solubility of fusion protein. Furthermore purification of target protein can be simplified by Protein A affinity chromatography. Especially, Fc-fusion can deliver corrective enzyme from the maternal to the fetal circulation during in utero ERT. In this study, N-terminus of GCD was fused to signal peptide of rice α-amylase 1A and C-terminus of GCD was fused to Fc region of human IgG1. Fc-fused GCD gene was subcloned into a pMYN409 vector which has a rice amylase 3D (RAmy 3D) promoter. This promoter is induced by sugar depletion. The expression vector was introduced into rice (Oryza sativa L. cv. Dongjin) calli by Agrobacterium-mediated transformation. The integration of gene into the rice calli was verified via genomic DNA PCR amplification. Fc-fused GCD in the cultured medium was detected by Western blot analysis and the quantity was analyzed by ELISA. 7 clones of calli were selected. The maximum yield of Fc-fused GCD was found to be 80 μg/L at day 10 after sugar depletion.