Marigold suspension cell culture as production system for recombinant DsRed and brazzein

Newly established Calendula officinalis suspension culture with low protease activity in the supernatant is transformed and produces high yields of recombinant proteins both in the cytosol and in the apoplast. A marigold (Calendula officinalis) suspension cell culture (CR1) was established from very young root tissues. Protease activity was checked in the cell culture supernatant (SN) and was found to be very low. To determine if CR1 could be a suitable production platform for recombinant proteins, a transformation was performed. Using Agrobacterium tumefaciens, CR1 was transformed with a construct containing two cassettes: one coding for a red fluorescent protein (DsRed) targeted to the endoplasmic reticulum and the other coding for the sweet protein brazzein, targeted to the apoplast. Suspension cultures were initiated from calli that exhibited strong DsRed fluorescence. A brazzein standard was prepared from the SN of one transformed CR1 line by purifying brazzein and subsequent quantification via tryptic digest and mass spectrometric comparison with a commercial brazzein-derived peptide of known concentration. This standard was used to determine the brazzein concentration in seven different transgenic CR1 transformation events by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the highest concentration measured at 24.5 µg/ml. Quantification of DsRed production in the cells of the same cultures showed up to 1322 µg/g fresh weight of DsRed (equivalent to 34.9% of total soluble protein). CR1 is potentially an excellent novel alternative to BY2 cells for the production of recombinant proteins.