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 OD₆₀₀ 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 Na₂EDTA; 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 Na₂EDTA; pH 7.5); Buffer B (20 mM Tris-HCl; 2M NaCl; 5 mM Na₂EDTA; 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 Na₂EDTA; pH 7.5) was added, followed by centrifuging at 5000×g to the final volume of 1 ml. This step was repeated twice.

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%); dH₂O 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.

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 DpnI 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 SalI and transforming into P. pastoris GS115. The recombinants were screened on MD plates without histidine and identified with PCR [16]. The expressions of the foreign proteins were identified with SDS-PAGE.

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 DpnI were incubated in 100 ml BMGY until the OD₆₀₀ 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 OD₆₀₀ 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.

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 OD₆₀₀ 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. OD₆₀₀ of all four cell cultures were measured every 2 h. When OD₆₀₀ 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 ₆₀₀ was measured every 6 h.

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.

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⁺, Mut^s) was provided by Biogrammatics (Carlsbad, CA, USA) and transformed by electroporation with 10 μg of linear plasmid

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

P. pastoris Bg11 cells were electroporated with 10 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 L fermentation. A single colony was grown in 100 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 1A), after which protein expression was induced with methanol at a concentration of 2 g/L. Every 24 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 1B), and total protein concentration was measured by Bradford (Figure 1C). The final dry cell weight for the fermentation was 300 g/L. After 120 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 2), where NoV VLPs elute in the peak between 0.1-0.2 M NaCl, as expected [34,35]. The elution fractions containing the NoV VLPs were pooled together and analyzed. The SDS-PAGE gel showed a band at 62 kDa corresponding to the molecular weight of the VLP protein. Western Blot using the anti-NoV serum also revealed a positive band at 62 kDa confirming the expression of the full-length VP1 protein (Figure 2). Protein concentration was over 1 g/L in the fermentation supernatant, and approximately 600 mg after protein purification.

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

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

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