Production of a recombinant miraculin protein in transgenic citrus cell suspension cultures

An efficient system for the production of a recombinant miraculin protein in transgenic citrus plant cells was developed. Miraculin is a taste-modifying protein that can transform a sour taste into a sweet taste. A miraculin gene containing a N-terminal signal peptide was introduced into navel orange (Citrus sinensis) callus cells by Agrobacterium-mediated transformation. Transgenic green color somatic embryos were generated on SIM medium containing 25 mg/L hygromycin and then embryogenic callus cell lines were produced from green color somatic embryos by using RCIM (Callus Reproduction Induction Medium) medium supplemented with 1 mg/L of GA3, 34 mg/L of adenine, 5% of sucrose, 500 mg/L of malt extract, and 0.2% of gelrite. The insertion of the miraculin gene in the transformants was confirmed by genomic-PCR, using specific primers of the miraculin gene. Cell suspension cultures were kept in MT liquid medium, pH 5.2, containing 5% sucrose. In order to overproduce the miraculin protein, the transgenic cells were transferred to fresh liquid suspension medium at 5% (weight of wet callus/volume of medium) density and were cultured at 15°C with rotation speed of 120 rpm. Recombinant miraculin expression and secretion in transgenic citrus suspension cells was confirmed by Western blot analysis. The recombinant miraculin protein was secreted into the culture medium and formed a disulfide-linked dimer similarly to the native miraculin with the taste-modifying activity. This production system could be a good alternative for production of the recombinant miraculin.