Editing Yeast Fermentation (section)

=== Deglycosylation of glycoproteins with Endo H-Pexpressed by ''P''. ''pastoris'' ===

Glycoprotein staining was performed using the Thermo Scientific Pierce Glycoprotein Staining Kit in accordance with the manufacturer’s recommendations (USA). An equal amount of each sample was loaded onto two 15% (w/v) oligosaccharide gels. After SDS-PAGE, one gel was stained with Coomassie Brilliant Blue G-250, and the other was stained using the Glycoprotein Staining Kit.
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The coding region of the NoV VP1 protein was cloned after the coding region for the α-factor and the expression was regulated under the methanol inducible AOX1 promoter. '''''P. pastoris Bg11'' (HIS<sup>+</sup>, Mut<sup>s</sup>) was provided by ''Biogrammatics'' '''(Carlsbad, CA, USA) and transformed by electroporation with 10&nbsp;μg of linear plasmid



Fermentation<br/>Fermentation was carried out as described previously<br/>[33,47]. A single P. pastoris colony was grown in 5 mL<br/>of YPD [1% (w/v) yeast extract, 2% (w/v) peptone, and<br/>2% (w/v) dextrose] overnight at 30°C. This culture was<br/>used to inoculate 100 mL of BMGY. The new culture<br/>was grown in a baffled flask at 30°C while shaking until<br/>it reached an OD 600 of ~20. This culture was used to in-<br/>oculate 1.5 L of fermentation medium.<br/>For the fermentation a Bioflo 3000 (New Brunswick<br/>Scientific, 2.5 L working volume) was used, interfaced with<br/>AFS-Biocommand Bioprocessing software version 2.6 (New<br/>Brunswick Scientific, Enfield, CT, USA) for data acquisition<br/>and control. The fermentation media consisted of 1.5 L of<br/>modified basal salts medium [0.23 gL −1 CaSO 4 · 2H 2 O,<br/>4.55 gL −1 K 2 SO 4 , 3.73 gL −1 MgSO 4 · 7H 2 O, 1.03 gL −1<br/>KOH, and 6.68 mL L −1 H 3 PO 4 , 5% (v/v) glycerol] and<br/>0.5 mL Antifoam 204 (Sigma, St. Louis, MO). For pH<br/>control, ammonium hydroxide [15% (v/v)] and phos-<br/>phoric acid [15% (v/v)] were used. PTM 1 trace salts<br/>(24 mM CuSO 4 , 0.53 mM NaCI, 19.87 mM MnSO 4 ,<br/>0.83 mM Na 2 MoO 4 , 0.32 mM boric acid, 2.1 mM CoCl 2 ,<br/>0.15 mM ZnCl 2 , 0.23 M FeSO 4 , and 0.82 mM biotin)<br/>were aseptically added at 4.35 mL L −1 after sterilization<br/>prior to inoculation. Temperature and pH were maintained<br/>at 30°C and 6, respectively. Dissolved oxygen (DO) was<br/>maintained at 40% of saturation and controlled by a DO-<br/>cascade agitation (with a maximum speed of 1000 rpm),<br/>supplemented with pure oxygen when needed, and mea-<br/>sured with an InPro6110/220 electrode (Mettler-Toledo<br/>GmbH, Germany).<br/>The batch phase was maintained until all the glycerol<br/>was depleted, as indicated by a sharp rise in DO (DO<br/>spike). At that point the culture was fed with methanol<br/>(100% methanol, with 12 mL PTM 1 L −1 ) and maintained<br/>at a constant concentration of 2 g/L. Every 24 hours,





''P. pastoris Bg11'' cells were electroporated with 10&nbsp;μg of linearized plasmid containing the secretion α-factor fused with the cDNA encoding the VP1 protein. Eight colonies were selected for further analysis. From these eight colonies, the colony with the highest protein expression, determined by western blot, was selected for a 1.5&nbsp;L fermentation. A single colony was grown in 100&nbsp;mL of BMGY as a preinoculum for the fermentation. Fermentation was initially grown in batch phase in medium with glycerol to increase biomass. The end of the batch phase was detected by a DO-spike (Figure&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/figure/Fig1/ <span>1</span>]A), after which protein expression was induced with methanol at a concentration of 2&nbsp;g/L. Every 24&nbsp;h an aliquot was taken from the bioreactor and dry cell weight was recorded and the culture supernatant was stored at 4°C. Protein expression was analyzed via SDS-PAGE gel (Figure&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/figure/Fig1/ <span>1</span>]B), and total protein concentration was measured by Bradford (Figure&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/figure/Fig1/ <span>1</span>]C). The final dry cell weight for the fermentation was 300&nbsp;g/L. After 120&nbsp;h of methanol induction, the extracellular medium was collected and the NoV VLPs were purified using ion-exchange chromatography, on a Sepharose Q XL column. A step gradient was used to elute the NoV VLPs. The chromatogram showed three major elution peaks (Figure&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/figure/Fig2/ <span>2</span>]), where NoV VLPs elute in the peak between 0.1-0.2&nbsp;M NaCl, as expected [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/#CR34 34],[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/#CR35 35]]. The elution fractions containing the NoV VLPs were pooled together and analyzed. The SDS-PAGE gel showed a band at 62&nbsp;kDa corresponding to the molecular weight of the VLP protein. Western Blot using the anti-NoV serum also revealed a positive band at 62&nbsp;kDa confirming the expression of the full-length VP1 protein (Figure&nbsp;[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174286/figure/Fig2/ <span>2</span>]). Protein concentration was over 1&nbsp;g/L in the fermentation supernatant, and approximately 600&nbsp;mg after protein purification.





P. pastoris transformation and selection of<br/>transformed cells. The selected recombinant<br/>plasmid<br/>pPICZ-scFv-EGFP-HPR<br/>was<br/>linearized with SacI. Transformation of the<br/>plasmid into yeast cells was carried out by<br/>electroporation according to the protocol<br/>suggested by Invitrogen and Biorad. A total of<br/>0.5 μg of linearized DNA was transferred into<br/>60 μl of competent cells of P. pastoris<br/>SMD1168H strain in an electroporation<br/>cuvette (2mm gap, BioRad). The cells and<br/>DNA mixture in the cuvette was incubated on<br/>ice for 2 minutes, then electroporation was<br/>carried out using Gene Pulser ΙΙ (Bio-Rad)<br/>with the following conditions: voltage (V) 2.5<br/>kV, capacitor (C) 50 μF, resistance (R) 200 Ω,<br/>and 5 ms pulse. Transformed colonies were<br/>grown and selected on YPDS zeocin selection<br/>medium (1% yeast extract, 2% peptone, 2%<br/>dextrose, 1 M sorbitol, 1.7% agar) containing<br/>100 μg/ml zeocin (Invitrogen).

Selection of P. pastoris transformants.<br/>Selection of P. pastoris transformants were<br/>carried out according to procedures given by<br/>Invitrogen. All of transformant colonies that<br/>grew on the selection medium were transferred<br/>into a new fresh YPD zeocin medium<br/>containing 100 μg/ml zeocin. The plates were

incubated at 30°C for 2 to 4 days. Stable<br/>transformants that grew on this medium were<br/>further transferred into YPD zeocin agar<br/>medium containing higher concentrations of<br/>zeocin (200, 500, and 1000 μg/ml,<br/>respectively) to acquire recombinant clones<br/>having multiple copies of target gene.<br/>Protein<br/>expression<br/>of<br/>P.<br/>pastoris<br/>transformants. Ten individual transformant<br/>colonies<br/>were<br/>cultured<br/>to<br/>evaluate<br/>recombinant protein expression according to<br/>manual instructions of Pichia expression kit<br/>(Invitrogen).<br/>Transformed<br/>and<br/>non-<br/>transformed P. pastoris cultivated in 2 ml of<br/>YPD medium (with or without zeocin) were<br/>incubated at 30°C and 250 rpm for 24 hours.<br/>These cultures were used to inoculate 25 ml of<br/>BMGY production medium (1% yeast extract,<br/>2% peptone, 100 mM potassium phosphate<br/>buffer pH 6.0, 1.34% YNB, 4×10 -5 % biotin,<br/>1% glycerol). Cultures were incubated at 30°C<br/>and 250 rpm for 24 hours. The cells were<br/>harvested aseptically by centrifugation before<br/>they were transferred into 25 ml BMMY<br/>induction medium (1% yeast extract, 2%<br/>peptone, 100mM potassium phosphate buffer<br/>pH 6.0, 1.34% YNB, 4×10 -5 % biotin, 0.5%<br/>methanol) with starting OD 600 =1.0. Cultures<br/>were incubated at 30°C and 250 rpm for 3<br/>days and methanol was added at 0.5% (v/v)<br/>into the culture every 24 hours. Cultures were<br/>harvested by centrifugation (10.000 rpm, 15<br/>min, 4°C). Cell biomass was stored at -20°C<br/>and the cell-free culture medium (supernatant)<br/>was stored at 4°C for further protein analysis<br/>with SDS-PAGE and immunoblotting (slot<br/>blot).
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