In silico codon optimization
hGM-CSF coding sequences were from Gene Bank (Accession M11220). In silico codon optimization was done using DNA 2.0 software with P. pastoris and S. cerevisiae codon tables and optimized parameters [8]. The optimization evaluation was done via http://www.gcua.schoedl.de with low (<20%) frequency codon usage displayed as hatched bars and very low (<10%) frequency codon usage as white bars.
Expression of rhGM-CSF in P. pastoris and S. cerevisiae
Recombinant P. pastoris and S. cerevisiae rhGM-CSF clones were expressed as described previously [9, 10]. The expression of the rhGM-CSF was analyzed by SDS-PAGE and immuno blot probed with an antibody against hGM-CSF (LifeSpan BioScience). Briefly, recombinant S. cerevisiae strain BY4742/hgm-csf was cultured in selective CSM-ura medium (6.7 g/L yeast nitrogen base with ammonium sulphate, 0.1 M sodium phosphate, supplemented with amino acids lacking uracil [11]) supplemented with 2% glucose until the OD600 of the culture was 0.5–1. The cells were then harvested and transferred into CSMG-ura medium (CSM supplemented with 20 g/L galactose) with shaking at 230 rpm at 30 °C. The pH 6.0 of each medium was stabilized by 0.1 M sodium phosphate buffer. The sample supernatants were collected after 72 h of induction and stored for further analysis.
Recombinant P. pastoris X33::hgm-csf was grown in 10 mL BMGY medium (2% peptone, 1% yeast extract, 0.34% yeast nitrogen base, 1% ammonium sulfate, 1% glycerol, and 0.4 mg/L biotin, buffered with 1/10 volume of pH 6.0 potassium phosphate buffer) at 30°C with constant shaking at 250 rpm until the culture reached an OD600 of 2–4. The cells were harvested and re-suspended in 10 mL BMMY (0.5% methanol is substituted for 1% glycerol in BMGY) with the same growth conditions. For induction, methanol was added every 24 h to a final concentration of 0.5%. After 72 h, culture supernatants were collected by centrifugation for further analysis.
Fermentation of recombinant P. pastoris and S. cerevisiae
Recombinant yeast S. cerevisiae strain BY4742/hgm-csf was batch-cultured in selective CSM(-ura) medium, pH 5.0 supplemented with 2% glucose until the culture reached an OD600 of 0.5 to 1, and then transferred into 3.18 L YP medium to a final OD600 of 0.5 in a 5 L LiFlus-GX fermenting vessel system (Biotron). Others growth parameters were temperature at 30 °C, aeration rate 1.5 vvm (through a filter), and agitation speed 720 rpm. Every 24 h, 40 g/L galactose was added at a rate of 6.7 mL/h. Samples were harvested at every 4 h and analyzed. Growth of the yeast cultures was monitored by optical density measurements at 600 nm. Protein secretion in the fermented medium was analyzed by SDS-PAGE and the protein concentration was determined using Bradford’s method [12]. The supernatant was stored at −20 °C for later purification.
For P. pastoris expression [13], a flask containing 150 mL BMGY containing 0.4 µg/mL biotin was inoculated from a frozen glycerol stock. The inoculum seed flask was grown at 30 °C, 250 rpm, and 24 h until OD600 = 2–6. A sterilized fermenter containing 2.5 L Fermentation Basal Salts medium supplemented with 4% glycerol and 11 mL PTM1 trace salts was prepared, the pH adjusted to 5.0 with ammonium hydroxide, the temperature set to 30 °C, agitation at 750 rpm and aeration to 5.0 vvm air. The culture in the inoculum seed flask was completely transferred into the fermenter in which the batch culture was grown for 24 h, until the glycerol was completely consumed. Glycerol feeding was initiated for about 4 h at a feed rate to 18.15 mL/h 50% w/v glycerol containing 1.2% v/v PTM1 trace salts. Methanol feeding was then initiated for 72 h at a feed rate of 100% methanol containing 1.2% v/v PTM1 trace salts as follows: 9 mL/h for 3 h; 18 mL/h for 3 h and then 27 mL/h. The supernatant was analyzed by SDS-PAGE every 4 h and the protein concentration was determined using Bradford’s method [12]. The supernatant was stored at −20 °C for later purification.
Ion exchange chromatography and hydrophobic interaction chromatography purification of recombinant rhGM-CSF
rhGM-CSF was purified using hydrophobic interaction chromatography. A HiPrep Phenyl Fast Flow column was equilibrated in buffer A [(NH4)2SO4 2 M, CH3COONa 20 mM, pH 5.0]. Approximately 20 mL of filtered supernatant was loaded onto the column at 1 mL/min. The column was then washed with 10 column volumes of buffer B. The bound protein was subsequently eluted with buffer B (Na2HPO4 10.14 mM; KH2PO4 1.76 mM; NaCl 136.89 mM; KCl 2.68 mM, pH 7.4) in a step-wise manner, ranging from 5, 10, 20, 40, 60 and 100% buffer B. The target fractions were identified by SDS-PAGE. The protein was eluted maximally at 10 to 20% buffer B.
rhGM-CSF was alternatively purified using anion exchange chromatography at pH 7.5. A HiTrap Q Sepharose Fast Flow column was equilibrated in 50 mM Tris-HCl. Approximately 20 mL of filtered and desalted supernatant was loaded onto the column at 1 mL/min. The column was then washed with 10 column volumes of 50 mM Tris-HCl. The bound protein was subsequently eluted in a step-wise manner with the NaCl concentration optimized over a range of 0–1 M NaCl in 50 mM Tris-HCl. The target fractions were identified by SDS-PAGE. The protein was eluted maximally at 0.1–0.2 M NaCl.
Bioactivity of purified rhGM-CSF
Purified rhGM-CSF from each yeast species was added to 1 × 105 cells/mL of TF-1 (a GM-CSF dependent cell line) in RPMI plus 10% FBS to obtain a final rhGM-CSF concentration ranging from 2 × 104 to 2 × 10−1 pg/mL. 100 µL of each combination was aliquoted into a 96-well plate and cultured for 72 h in a 5% CO2 incubator. The proliferation of TF-1 cells was measured by using a CCK-8 kit at OD 450 nm according to the manufacturer’s instructions. Crude supernatants from P. pastoris and S. cerevisiae served as controls. LU (Laboratory unit) was calculated as follows:
$${\text{LU}}\,({\text{units}}/{\text{mg}}) = \frac{{1 \times 10^{9} }}{{{\text{ED}}50}}$$
