Hybanthus enneaspermus subsp. suspension

Doffek, Benjamin, Huang, Yvonne, Huang, Yen-Hua, Chan, Lai Yue, Gilding, Edward K., Jackson, Mark A. & Craik, David J., 2022, Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming, Phytochemistry (113053) 195, pp. 1-10 : 8

4.9. Scale up of H. enneaspermus suspension culture

The cultivation parameters were 26 ◦ C, 16:8 light-dark cycle, 20 μmol m ⁻ 2 s ⁻ 1 photon density, rocking angle of 10 ◦, 20 rpm and a gas flow rate of 0.1 L min 1 air per 1 L culture volume (supplemented with 5% (v/v) CO 2). The cells were harvested after 20 days at maximum cell density in the stationary phase. Suspension cells were pelleted by centrifugation at 8000× g for 20 min and subsequently lyophilized overnight. The dry weight was recorded, and peptides extracted using 50 mL of solvent (50% acetonitrile 1% formic acid) per gram dry weight with insoluble material removed by centrifugation (5000× g, 20 min), and filtration (0.45 μm). Peptide containing extract was then transferred into a round bottom flask then lyophilized. After re-dissolving in 25 mL of 50% acetonitrile, 1% formic acid the sample was diluted to ~5% acetonitrile, 1% formic acid and loaded onto a C18 solid phase extraction (SPE) column. Fractions were eluted using 2 SPE column volumes of 0, 10, 20, 30, 50, 60 and 80% acetonitrile, 1% formic acid, and analyzed for putative cyclotides using MALDI-TOF-MS. Fractions containing cyO2 were further purified using HPLC initially with a semi-preparative column (flow rate 3 mL per minute; 1% acetonitrile gradient) and later an analytical scale column (flow rate 1 mL per minute; 1% acetonitrile gradient) and checked for purity by LC/MS and structure using NMR.