Yeast transformation and induction

While the focus of this work was FAS, which is constitutively expressed in yeast³³, our initial aim was to use yeast to express and purify VLPs composed of hepatitis B virus tandem core proteins²⁷ with a SUMO-binding affimer²⁸ inserted into the major immunodominant region (MIR) (construct available upon request) for structural characterisation. As such, VLP expression was induced in the transformed yeast prior to purification, as described previously³⁷. Briefly, glycerol stocks known to exhibit good levels of expression were cultured in yeast extract-peptone-dextrose (YPD), supplemented with 50 µg/mL ampicillin, at 28 °C, 250 rpm for 48 h. 2 ml of the high-density starter culture was then transferred to 200 mL YPD and incubated for another 24 h under the same conditions. To induce expression of VLPs, cells were pelleted (1500 × g, 20 min) and resuspended in 200 mL yeast extract-peptone-methanol (YPM; 0.5% [v/v] methanol) for a further 48-h incubation, with methanol added to a final concentration of 0.5% (v/v) 24 h post-induction. Cells were subsequently collected by pelleting at 2000×g (20 min), resuspended in 30 mL breaking buffer (50 mM sodium phosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM EDTA, 5% glycerol, pH 7.4) and stored at − 20 °C prior to purification.