Editing Yeast Fermentation

Cells for Pichia Expression

GS115 P. pastoris cells from Invitrogen



Recombinant ''P''. ''pastoris'' GS115 bearing the target gene in its genome was incubated in 100 ml of BMGY until the OD<sub>600</sub> value reached approximately 20. All cells were harvested by centrifugation and transferred to 25 ml BMMY. Two hundred and fifty microliter of 1% (v/v) methanol was added every 24 h to induce target protein expression. Approximately 1 ml of cell culture was collected every 24 h and centrifuged at 6,000×g for 5 min to remove cells. After 144 h, an equal volume of each supernatant (15 μl) was loaded onto a 12% (w/v) polyacrylamide gel for SDS-PAGE, followed by staining with Coomassie Brilliant Blue G-250. The total protein concentration in each supernatant was measured using the Micro-BCA Protein Assay Reagent (Pierce, USA).

After induction with methanol for 120 h, the target protein was purified by centrifuging approximately 20 ml of BMMY cell culture at 6000×g for 5 min. The supernatant was then collected and dialyzed with a Millipore 10-kDa cut-off membrane at 4°C to remove ions and salts. The sample was resuspended in 15 ml of Buffer A (20 mM Tris-HCl; 5 mM Na<sub>2</sub>EDTA; pH 7.5). The sample was centrifuged at 5000×g to the final volume of 1 ml. This step was repeated twice. The 1-ml solution was added to the diethylaminoethyl dextran gel (DEAE) FF and washed under the following condition: Buffer A (20 mM Tris-HCl; 5 mM Na<sub>2</sub>EDTA; pH 7.5); Buffer B (20 mM Tris-HCl; 2M NaCl; 5 mM Na<sub>2</sub>EDTA; pH 7.5), with the velocity of 0.5 ml/min. The target protein was eluted with 1.4 M NaCl. The elute (about 2 ml) was added to a Millipore 10-kDa cut-off membrane, and 13 ml of Buffer C (20 mM Tris-HCl; 50 mM NaCl; 5 mM Na<sub>2</sub>EDTA; pH 7.5) was added, followed by centrifuging at 5000×g to the final volume of 1 ml. This step was repeated twice.
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=== Analyzing the enzymatic activity of the recombinant Endo H ===

The hydrolytic activity of Endo H from ''P''. ''pastoris'' and commercial source (heterlogously expressed in ''E''. ''coli'') was compared with carboxypeptidase Y (CPY) and human erythropoietin (EPO) as substrates. About 10 U of Purified recombinant Endo H or commercial Endo H was added to 10 μg of CPY (Sigma, USA) and 10 μg of EPO (Cusabio, China), and incubated in proper buffer at 37°C for 1 h. Meanwhile, 10 U of PNGase F (NEB, USA) was used to deglycosylate EPO under the same condition. All samples were loaded for SDS-PAGE.

RNase B (10 μg) was denatured with 1× Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After this treatment, 1 μl of reaction buffer (50 mM sodium citrate, pH 5.5) was added along with 1 μl of the dilutedrecombinant Endo H-P (5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%); dH<sub>2</sub>O were added to the final volume of 10 μl. The mixture was incubated at 37°C for 1 h. SDS-PAGE was used to detect the hydrolyzed products. One unit (U) of Endo H was defined as the amount of enzyme required to remove >95% of carbohydrates from 10 μg of denatured RNase B in 1 h at 37°C in a total reaction volume of 10 μl (10NEB units = 1 IUB milliunit). The optimum temperature was determined by incubating 10 μg denatured RNase B with 1 μl reaction buffer and in a final volume of 10 μl at the proper temperature for 1 h. The optimum pH was determined by incubating 10 μg denatured RNase B with 1 μl reaction buffer adjusted to a series of pH values from 5.0 to 7.5 in a final volume of 10 μl at 37°C for 1 h. The experiments were performed in triplicate.
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=== Construction and enzyme activity of the substrates ===

Recombinant ''P''. ''pastoris'' strains expressing endo-1, 4-β-mannosidase from ''Aspergillus niger'', DNase I from ''Bos taurus'', phytase from ''E''. ''coli'' and ''Dpn''I from ''Diplococcus pneumoniae'' were constructed in our lab. The recombinant strains expressing substrates were generated with the similar strategy as Endo H-P expressing ''P''. ''pastoris''. The coding sequences of these genes were optimized according to the codon usage preference of ''P''. ''pastoris'' and synthesized with overlapping PCR. The newly synthesized ORFs were sequenced and inserted into pHBM905A vectors, followed by digesting with ''Sal''I and transforming into ''P''. ''pastoris'' GS115. The recombinants were screened on MD plates without histidine and identified with PCR [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362766/#pone.0120458.ref016 16]]. The expressions of the foreign proteins were identified with SDS-PAGE.
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=== Deglycosylation of recombinant glycoproteins expressed in ''P''. ''pastoris'' using the recombinant Endo H ===

For the post-fermentation treatment, the recombinant ''P''. ''pastoris'' strains producing endo-1, 4-β-mannosidase, phytase and ''Dpn''I were incubated in 100 ml BMGY until the OD<sub>600</sub> value reached approximately 20. All cells were harvested by centrifugation, transferred to 25 ml BMMY and then induced with 1% (v/v) methanol for 144 h. The cell culture supernatant of the ''P''. ''pastoris'' producing recombinant Endo H-P was prepared using the same procedure. The supernatant containing each glycoprotein was mixed with that containing recombinant Endo H-P at a ratio of 10:1 and incubated at 37°C for 1 h.

For co-fermentation, the recombinant ''P''. ''pastoris'' strains expressing DNase I and phytase were separately inoculated in 100 ml BMGY and incubated until the OD<sub>600</sub> value reached approximately 20. All cells were collected from these two samples and transferred into two flasks containing 25 ml BMMY in each. Cells from a 10-ml culture of recombinant Endo H-P-expressing ''P''. ''pastoris'' prepared under the same conditions were collected and added to both flasks, which were then induced with 1% (v/v) methanol for 144 h.
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=== The growth curve of the recombinant strains under the co-fermentation condition ===

The effect of Endo H-P to the growth rate of the recombinant ''P''. ''pastoris'' GS115 expressing phytase of ''E''. ''coli'' was studied. ''P''. ''pastoris'' strains expressing Endo H-P and phytase and ''P''. ''pastoris'' GS115 strain bearing pHBM905A plasmid were incubated in 10 ml of BMGY separately until the OD<sub>600</sub> reached approximately 1.0, then 1ml of the cultures were injected into 100 ml of BMGY separately. Meanwhile, 100-μl culture of the recombinant Endo H-P-expressing ''P''. ''pastoris'' and 900-μl culture of the recombinant phytase-expressing ''P''. ''pastoris'' were mixed and transferred into 100 ml of BMGY. OD<sub>600</sub> of all four cell cultures were measured every 2 h. When OD<sub>600</sub> of the cell cultures reached about 20, all cells were harvested by centrifugation and transferred to 50 ml BMMY. All four cell cultures were induced with 1% (v/v) methanol and OD <sub>600</sub> was measured every 6 h.
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=== 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).
[[Category:Protocols]]