Development of optimal fermentation expression systems for recombinant proteins: a thesis submitted in partial fulfilment of the requirements for the degree of Masters in Technology at Massey University

Abstract

This research set out to maximise the titre of four recombinant protein products (i.e. Eg95 vaccine antigen against Echinococcus granulosus a aspartyl protease inhibitor homologue, Aspin; a secreted cytokine granulocyte colony stimulating factor (G-CSF); a secreted gonadotropin ovine follicle stimulating factor (oFSH)) and develop parameters for the expression of those proteins in a small scale stirred tank biorcactor. Production of Eg95 as inclusion bodies in E. coli was influenced by the medium, feeding strategy, induction timing and dissolved oxygen concentration. Expression was greatest using the medium Terrific Broth. Higher Eg95 titres were favoured using exponential feeding, a low dissolved oxygen concentration and with cells induced in mid-exponential growth. A maximum titre of 1.73 g/L of Eg95 was produced in a fed-batch fermentation controlled at 37°C, pH 7.0 and 30% dissolved oxygen. Induction with 0.1 mM of IPTG added four hours after inoculation, was optimal. The maximum titre attained, was a 360% improvement on fermentations prior to this research. Aspin was used to investigate the culture conditions for maximizing the production of soluble protein in E.coli. Soluble Aspin production was favoured at low expression rates. A volumetric titre of 0.220 g/L of soluble Aspin was attained in batch fermentation by inducing with 2 g/L of L-arabinose, with the temperature reduced from 37°C to 23°C and by maintaining a low dissolved oxygen (DO) concentration. This yield was relatively high compared to previous reports [1-3]. G-CSF production in the yeast Pichia pastoris was influenced by the medium, pH and methanol-to-cell ratio. A maximum titre of 0.028g/L of G-CSF was produced in shaker flasks of enhanced yeast extract Hy-Soy dextrose medium (YEHD), maintained at 200 rpm, 30°C, pH 6.0 and with 1% (v/v) methanol fed per day. Cells were resuspended to an optical density of 8 prior to induction. No improvement in G-CSF was achieved in the fermenter, likely due to an inhibition by toxic materials. The optimised shaker flask yield was consistent with previous reports [4-6]. Production of oFSH in insect cells was influenced by the cell density at inoculation and rate of agitation. 0.001 g/L of oFSH was produced in shaker flasks inoculated at a density of 1 x 106 cells/mL, cultured at 27°C and agitated at 140rpm. This represented an improvement over previous yields[7].