|Home | About | Journals | Submit | Contact Us | Français|
Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. Pichia pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, Pichia pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL-3 from the mixture of expressed glycosylated and non-glycosylated porcine IL-3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL-3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in Pichia pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.
Hematopoietic stem cell (HSC) transplantation is a therapeutic strategy used for hematologic malignancies, immunodeficiency states, nonmalignant hereditary and acquired hematologic diseases, as well as inherited metabolic disorders [1, 2]. HSCs can be mobilized from the bone marrow into peripheral blood and collected by apheresis. This method of peripheral blood stem cell collection is frequently used clinically for obtaining HSCs for allogenic and autologous HSC transplantation. Currently, injection of granulocyte colony-stimulating factor (G-CSF) is the clinical standard for mobilization of human HSC . Porcine IL3 combined with porcine stem cell factor (SCF) has been shown to be more effective at stem cell mobilization than G-CSF in our preclinical miniature swine model. Pichia pastoris expressed porcine IL3 has been used for porcine bone marrow stem cell mobilization in an allo-hematopoietic cell transplantation model and pig-to-primate xeno- hematopoietic cell tranplantation model in our lab for many years [3, 4, 5, 6, 7, 8]. However, since the N-glycosylation mechanism is different between yeast and mammalian cells , Pichia pastoris expressed high-mannose glycoprotein porcine IL3 could lead to rapid clearance in vivo . This was avoided by use of the non-glycosylated porcine IL-3 which was separated from the mixture of expressed glycosylated and non-glycosylated porcine IL-3. This process was very inefficient and lead to poor yields following purification.
In this study, we replaced all four potential porcine IL3 N-glycosylation sites with alanines. The non-N-glycosylated porcine IL3 was expressed and purified in Pichia pastoris. In vivo mobilization study demonstrated that removal of these glycosylation sites did not result in a decrease in the mobilization function
Using the published porcine IL3 gene sequence , the non-N-glycosylated porcine IL3 gene (Figure 1) was synthesized using the Pichia pastoris preferred codons . Ten primers (Table 1) were designed to cover the full length of the non-N-glycosylated porcine IL3 gene (121 aa). There was an average 21 base overlap between neighboring primers. 10 pmol of the first and the last primer, and 2 pmol rest of the primers were used. PCR was performed at 95°C for 5 min, 25 cycles of 95°C for 30 sec, 55° for 30 sec, 72° for 1 min and an extension at 72° for 10 min. The PCR products were analyzed with 1% agarose gel electrophoresis, and the DNA fragment with the predicted size was cut out and extracted with QIAquick Gel Extraction Kit. The DNA was digested using XhoI and EcoRI, cloned into pwPICZalpha  and then sequenced. To facilitate the downstream purification, six histidines (6×His tag) were added to the C-terminus by PCR amplification using primers pIL3NGF and pIL3NGR (Table 1). The PCR product was digested with XhoI + EcoRI and cloned back into pwPICZalpha and sequenced to generate the non-N-glycosylated porcine IL3 construct carrying 6×His tag in its C-terminus.
5–10 µg of the above constructed plasmid DNA was linearized by SacI digestion for 3 h at 37°C, treated with Qiagen PCR purification kit, and transformed into Pichia. pastoris strain X33 using the Gene Pulser Xcell Electroporation system (Bio-Rad). Cells were spread on YPD agar plates (1% Bacto™ yeast extract, 2% Bacto™ peptone, 1.5% Bacto™ agar, 2% dextrose) containing 100 µg/mL of Zeocin and incubated at 30°C for 3–4 days. Six colonies were randomly picked and cultivated in test tubes containing 5 mL YPD (1% Bacto™ yeast extract, 2% Bacto™ peptone, 2% dextrose) at 30°C at 250 rpm for 24 h as growth phase I, then in YPG (1% Bacto™ yeast extract, 2% Bacto™ peptone, 1% glycerol) at 30°C at 250 rpm for another 24 h as growth phase II. The cultures were induced in 2 mL BMMYC (1% Bacto™ yeast extract, 2% Bacto™ peptone, 100 mM potassium phosphate, pH 7.0, 1.34% yeast nitrogen base without amino acids (MP), 4 × 10−5 % biotin, 0.5% methanol and 1% Bacto™ casamino acids) for 48 h at 25°C at 225 rpm. 0.5% methanol was added at the beginning and end of the day to sustain the methanol level. The culture supernatants were analyzed using 12% NuPAGE SDS gel under non-reducing conditions.
One clone was selected and cultivated in shake-flasks (scaled up from the above described small tube expression) for downstream purification. 100 units/mL of penicillin and 100 µg/mL of streptomycin were added to suppress bacterial contamination. The expressed protein in the supernatant was harvested by centrifugation at 3000 rpm at 4°C for 10 min prior to protein purification.
Ni-Sepharose™ 6 fast flow resin (GE healthcare) was packed in a XK16/20 column (GE healthcare) for the first step purification. The column was equilibrated with 20 mM sodium phosphate pH 7.4, 0.5 M NaCl, and 5 mM imidazole (10 CV). The sample was prepared by adding 0.5 M NaCl, 20 mM sodium phosphate pH 7.4, and 5 mM imidazole and filtering through crepe fluted filter paper (VWR) before being loaded onto the equilibrated column. The column was washed using 20 mM sodium phosphate pH 7.4, 0.5 M NaCl, and 5 mM imidazole (8 CV). The bound proteins were eluted with 20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 250 mM imidazole in eight fractions, which were analyzed using 12% NuPAGE® Bis-Tris gel stained with GelCode Blue stain reagent (Thermo Scientific). The fractions containing protein of interest were pooled and dialyzed against 20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 5% glycerol at 4°C in a 3.5 kDa cut off Spectra/Por® membrane tubing (Spectrum labs). The dialysis buffer was replaced once.
Strong anion exchange resin Poros® 50 HQ (Applied Biosystems) in a XK16/20 column (GE Healthcare) was used for the second step purification. The column was equilibrated with 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 5% glycerol (10 CV). The dialyzed sample was loaded, then the column was washed with 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 5% glycerol (8 CV). The bound protein was eluted with 50 and 100 mM sodium borate in 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 5% glycerol into eight fractions, respectively. The purification fractions were analyzed using 12% NuPAGE SDS gel. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific).
Protein samples were separated by electrophoresis using NuPAGE 12% Bis-Tris Gel and the gel was electro-transferred at 35 V onto nitrocellulose membrane filter paper using 1x NuPAGE® transfer buffer (Invitrogen). The membrane was thereafter blocked in 5% blotting grade blocker non-fat dry milk (Bio-Rad) in 1xPBS, 0.02% Tween 20 for 1 h with shaking and washed once with 1xPBS, pH7.4, 0.02% Tween 20 at room temperature with shaking. The non-N-glycosylated porcine IL3 was detected with mouse anti-His monoclonal antibody (1:500) (Invitrogen) and rat anti-mouse IgG-HRP (1:1000) (Invitrogen) in 5% non-fat dry milk in 1xPBS, 0.02% Tween 20. Detection of the proteins was done by TMB membrane peroxidase substrate (KPL Cat#:50-77-02), and color development was stopped with dH2O.
Massachusetts General Hospital (MGH) Major Histocompatibility Complex (MHC)-defined miniature swine ranging in weight from 30–60 kg were used for this study. Mutated non-N-glycosylated porcine IL3 was compared to the original porcine IL3 developed by Biotransplant Inc  as control. This control porcine IL3 was named as porcine IL3-BTI to distinguish it from the non-N-glycosylated porcine IL3. Animals were treated for 5 to 7 days with a weight-based dose of recombinant porcine IL3 and porcine stem cell factor (0.1mg each of porcine IL3 and porcine SCF per kg recipient body weight for first 30 kg, 0.05 mg/kg for each additional kg). The porcine IL3 was used in combination with porcine SCF to keep our established mobilization protocol consistent. All animals were pre-medicated using diphenhydramine (2–4 mg/kg, IM or IV) 30 min before each porcine IL3 injection. PBMC (peripheral blood mononuclear cells) collection was achieved by leukapheresis (COBE BCT Inc., Lakewood, Colorado, USA) starting after 5 days of cytokine injection.
The Cobblestone area forming cell (CAFC)  assay measures a spectrum of hematopoietic cells and allows committed precursor cells (day 7 to week 8) to be separated from more primitive HSC with long-term repopulating ability (day 28–35 CAFC). EUR-FLS-72/1C5A stromal cells originally derived from day -12 fetal livers of CBA×C57BL/10 F1 mice  were overlaid with various dilutions of PBMC to allow limiting dilution analysis of the precursor cells forming hematopoietic clones under the stromal layers (16 wells per dilution with eight 2-fold dilution steps). The cells were cultured at 37°C, 5% CO2 for 8 weeks in CAFC medium [Iscove’s modified Dulbecco’s medium with GlutaMAX-1 (Invitrogen, Carlsbad, CA) supplemented with 25 ng/mL of porcine SCF, 2 ng/mL of porcine IL3, gentamicin (10 mg/mL), 2-mercaptoethanol (1 × 10−4 M), 12.5% fetal bovine serum, 12.5% horse serum, and hydrocortisone (1 × 10−6 M)] with weekly replacement of 80% of the medium. The percentage of wells with at least 1 phasedark hematopoietic clone of at least 5 cells (i.e. cobblestone area) beneath the stromal layer was determined weekly (or on week 5 to week 8). Frequencies of the week 8 CAFC subsets were calculated with L-cal statistical software (Stem Cell Technologies Inc.).
As shown in Figure 1 the four potential N-glycosylation sites (N24, N30, N43, N64) in the porcine IL3 gene were replaced with non-polar amino acid alanines. We expected that the four replacements would not influence the porcine IL3 mobilization function. The codon-optimized non-N-glycosylated porcine IL3 gene carrying 6x His tag (127 aa) in the C-terminus was synthesized (GenBank accession number: BankIt1492785 IL3 JQ028875). The 6xHis tag in the C-terminus was added to facilitate the downstream purification.
The non-N-glycosylated porcine IL3 carrying 6xHis-tag in the C-terminus was expressed in shake flask cultures. Western blotting analysis confirmed the expression using mouse anti-His monoclonal antibody (data not shown). The secreted non-N-glycosylated porcine IL3 was captured directly by Ni-Sepharose 6 fast flow resin through its 6xHis-tag in the C-terminus mainly in the 2nd and 3rd fractions eluted (Figure 2A). The eluted fractions from the capturing step were pooled, concentrated down with Centricon Plus-70 (5 kDa cut off) and dialyzed to remove the salts for the second step purification. Based on the isoelectric point value (6.11) strong anion exchange resin Poros 50 HQ was chosen for the second step purification. In the second step purification sodium borate was applied to separate the non-N-glycosylated porcine IL3 from the glycosylated yeast host protein and aggregates . As shown in Figure 2B we obtained the pure non-N-glycosylated porcine IL3 after the two-step purification. SDS-PAGE and Western blot analysis with anti-6xHis mAb for the final product is shown in Figure 3. After mutating the 4 potential N-glycosylation sites and adding the 6xHis tag in the C-terminus, the binding to the strong anion exchange resin Poros 50HQ improved dramatically under pH 8.0 which facilitated our second step purification. The absence of the N-glycosylation improved the expression and purification significantly. Prior to mutating the N-glycosylation sites, the expression level was ~10 mg/L before purification and 6.6 mg/L after two-step purification. After the mutation, the expression level improved to ~90 mg/L before purification and 75 mg/L after two-step purification.
To assess the ability of mutated non-N-glycosylated porcine IL3 to mobilize stem cells into the peripheral blood, we injected the non-N-glycosylated porcine IL3 or the control porcine IL3-BTI intramuscularly into MGH MHC-defined miniature swine. The frequency of CAFC activity following non-N-glycosylated porcine IL3 mobilization was comparable to the frequency of CAFC activity following porcine IL3-BTI control mobilization (Figure 4, Table 2). These results demonstrated that the non-N-glycosylated porcine IL3 can replace the porcine IL3-BTI as a mobilization reagent in vivo. The in vivo mobilization study also demonstrated that the 6xHis tag added in the C-terminus did not influence the mobilization function. Since the mobilization dose requirement is high (~0.7 mg/kg), cost-effectiveness, high production level, high purity, easy purification is needed. As presented in this study, the non-N-glycosylated porcine IL3 met all of the above requirements.
This work was supported by National Institutes of Health (R01AI084657-02 to CAH); and Dana Farber/Harvard Cancer Center Core development grant.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.