Centriprep 50ym

The chimera gB vaccine candidate, composed of PH-like domain 1 from BoHV-1 and PH-like domain 2 from BoHV-5 (hereafter DgB), was expressed in Komagataella phaffii GS115 and produced as previously described [21 (link),22 (link),23 (link)]. After induction phase, the recombinant protein was concentrated from the culture supernatant by using Centriprep 50YM (Millipore, Burlington, MA, USA) and purified by affinity chromatography using both HisTrap™ HP 1 mL columns pre-packed with pre-charged Ni Sepharose™ and the ÄKTAprime™ Automated Liquid Chromatography system (GE Healthcare, Chicago, IL, USA). Protein concentration was determined by Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA USA), according to the manufacturer’s instructions. The chimera DgB was lyophilized and used for the different vaccine formulations.

The cloning of the BoHV-5 (strain SV507/99) glycoprotein D into P. pastoris strain KM71H Mut^S expression system, as well as the expression of the recombinant glycoprotein D (rgD5) were previously described [22 (link)]. Briefly, the recombinant clone was inoculated in culture flasks containing buffered glycerol-complex (BMGY) medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 0.00004% biotin, 1% glycerol, 100 mM potassium phosphate pH 6.0) and incubated in orbital shaker for 24 h at 28°C with agitation speed of 150 rpm. The cells were harvested and resuspended in 1/10 (10%) of the original culture volume of buffered methanol-complex (BMMY) medium (BMGY medium with 0.5% methanol in replacement of 1% glycerol). To induce expression, 1% of 100% methanol were added every 24 h, and cells were kept in the same growth conditions described above for 72 h. Cells were then harvested and the collected supernatant concentrated with Centriprep 50YM (Millipore) device at 1,500 x g for 10 min at 20°C. The rgD5 was then purified by affinity chromatography using both HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose and the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare).