This includes production in the 1-L and 5-L scales in the yeast, X33 strain was transformed with expression plasmid pPICZA containing RBD219-N1C1 coding DNA, and one transformed colony with high expression of recombinant RBD219-N1C1 protein (Chen et al. solitary colony from your respective plate and incubated at 30 C with constant shaking (225 rpm) until the OD600 reached 9.3. Finally, the cell tradition was mixed with plant-derived glycerol to a final concentration of 20% and aseptically aliquoted (1 mL each) into 1.2-mL cryovials. For long-term storage, the cryovials were stored at ?80 C. Fermentation One vial of the SARS-CoV-2 RBD219-N1C1 RCB was used to inoculate a 0.5-L buffered minimal glycerol (BMG) medium inside a 2-L baffled shake flask. The shake flask tradition was cultivated at 30 C and 225 rpm until an OD600 of 5C10. For 1-L fermentations, this seed tradition (20C40 mL) was inoculated into 0.4 L of sterile basal-salt medium (BSM) (pH 5.0; BSM: 18.2 g/L potassium sulfate, 14.9 g/L magnesium sulfate heptahydrate, 4.13 g/L potassium hydroxide, 0.93 g/L calcium sulfate Esr1 dehydrate, 26.7 mL/L of 85% phosphoric acid, and 40 g/L glycerol) or low-salt medium (LSM) (pH 5.0; LSM: 4.55 g/L potassium sulfate, 3.73 g/L magnesium sulfate heptahydrate, 1.03 g/L potassium hydroxide, 0.23 g/L calcium sulfate dehydrate, 10.9 mL/L of 85% phosphoric, and 40 g/L glycerol) to a starting cell density (OD600) of 0.5. Fermentation was carried out using a Biostat Qplus bioreactor having a 1-L vessel (Sartorius Stedim, Guxhagen, Germany). For SEL120-34A HCl 5-L runs, the seed tradition (125C250 mL) was inoculated into 2.5 L of LSM, and fermentation was carried out inside a CelliGen 310 bioreactor having a 7.5-L vessel (Eppendorf, New York, USA), controlled from the Eppendorf Bio Command software. Cell development was continued at 30 C having a dissolved oxygen (DO) set point of 30%. After 19 2 h of growth, a dissolved oxygen spike was observed on the tendency chart, which shows glycerol depletion. A fed-batch was initiated with 50% glycerol at a feed rate of 15 mL/L/h for 6 h to further expand biomass. During the last hour of the fed-batch phase, pH was modified to 6.5 SEL120-34A HCl using 14% NH4OH, while the temperature was modified to 25 C. When a glycerol fed-batch was not included in the fermentation process, the pH and temp were modified to the desired value during the 1st hour of induction. After the fed-batch phase, methanol induction was initiated; the total induction time was approximately 68C72 h. Biomass was eliminated by centrifugation at 12,227for 30 min at 4 C before the supernatant was filtered through 0.45-m polyethersulfone (PES) filters stored at ?80 C until purification. Purification overview of three processes The fermentation supernatant (FS) was removed from ?80 C and thawed at 22 C for 4C6 h. Three purification processes were performed with 1-L FS aliquots (Fig. ?(Fig.1b).1b). In process 1, the RBD219-N1C1 protein was captured from your FS using hydrophobic connection chromatography (HIC), concentrated by ultrafiltration/diafiltration (UFDF), and polished using size exclusion chromatography (SEC). In process 2, the RBD219-N1C1 protein was captured using HIC, buffer-exchanged (UFDF), and polished using anion-exchange chromatography (AEX). Finally, in process 3, the FS was buffer-exchanged using UFDF before the target protein was captured using cation-exchange chromatography (CEX), buffer-exchanged (UFDF), and polished using AEX. Open in a separate windowpane Fig. 1 Fermentation (a) and purification (b) circulation diagrams. Three purification processes performed are demonstrated in different colours. The color plan remains consistent throughout all numbers. UFDF, ultrafiltration and diafiltration; HIC, hydrophobic connection chromatography; SEC, size exclusion chromatography; TFF, tangential circulation filtration; CEX, cation exchange chromatography; AEX, anion exchange chromatography UFDF (ultrafiltration and diafiltration) Two types of products were utilized for UFDF, a centrifugal concentrator, and a flat sheet membrane, depending on the target volume. For process 1, Amicon centrifugal concentrator, having a 10 kDa molecular excess weight cutoff (MWCO) (MilliporeSigma, Burlington, SEL120-34A HCl USA) was used to concentrate the HIC elution pool (2050at 4 C). This allowed concentration to the small volume needed for SEC. For process 2, a flat sheet Pellicon XL Cassette having a Biomax 5 membrane (5 kDa MWCO) and a Labscale TFF System (MilliporeSigma, Burlington, USA) were used to concentrate the.