Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using difference in gel electrophoresis

초록

Difference in gel electrophoresis (DIGE) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. It provides accurate quantitative and reproducible differential expression values for proteins in several samples. Recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (rhCTLA4Ig) was produced in the transgenic rice suspension cell cultures using ？?amylase promoter system. This system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. In this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-D DIGE. Each sample from different conditions and internal standard were labeled with N-hydroxy succinimidyl ester-derivatives of Cy2, Cy3 and Cy5 dyes and run within a single DIGE gel. Using DeCyderTM software, 2,218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhCTLA4Ig with normalization method improving data distribution. A pooled sample mixture for the picking gel was prepared with Deep Purple staining and analyzed with mass spectrometry. In addition, the intracellular rhCTLA4Ig spots were identified with Western blot analysis using goat anti-human IgG (Fc) antibody after DIGE gel was transferred to PVDF membrane.