Strains, plasmids, antibodies and equipment
Pichia pastoris strain GS115, Escherichia coli strain DH5α, and expression vector pPIC9 were purchased from Invitrogen (Life Technologies). MDBK and Daudi cells were obtained from Wuhan Boster. The plasmid pPIC9K-Kex-IFNα2b-Fcγ1 with the coding gene of fused human IFN-α2b and the wild-type human IgG1 Fc fragment was constructed and preserved in our laboratory . WISH cells, the conventional IFN-α and the anti-IFN-α monoclonal antibody were obtained from Anke Biotechnology Group. HPR-conjugated anti-mouse antibodies were purchased from Cell Signaling. The 14-L fermenter (New Brunswick BioFlo115) used in pilot-scale fermentation was obtained from Eppendorf. A flex stand system with 0.45-μm hollow fiber membrane filtration cartridges and an AKTA Avant system with MabSelect and S-200 HR columns were obtained from GE Healthcare.
Molecular design and cloning
The fusion protein IFN-α/Fc-WT was secreted into culture medium as the active form of disulfide-linked homodimer with a single N-glycosylation modification in the IgG1 Fc region in each molecule. Overlap extension PCR was used for site-specific mutagenesis of the glycosylation site on the IgG1 Fc fragment and for the insertion of a flexible GS linker between the C-terminus of IFN-α and the N-terminus of the IgG1 Fc fragment.
The primer pairs (see Additional file 1: Table S1) α-Fw and SC-Rv, SC-Fw and M-Rv, and M-Fw and Fc-Rv were used to amplify three parts of the fused coding sequence of the IFN-α/Fc-SC monomer on the template plasmid pPIC9K-Kex-IFNα2b-Fcγ1 (pIFN-α/Fc-WT), which was used for expression of the fusion protein IFN-α/Fc-WT. The PCR procedure was as follows: 94 °C for 5 min; 32 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 60 s; and 72 °C for 10 min. The primer pair α-Fw and Fc-Rv was used to splice the three amplified parts to obtain the complete fused IFN-α2b and the IgG1 gene. The PCR procedure was as follows: 94 °C for 5 min; 32 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 120 s; and 72 °C for 10 min. This newly amplified gene encoding a single-chain IFN-α2b and the IgG1 Fc fragment-fused protein contained a mutated glycosylation site on the IgG1 Fc fragment (297 N to Q) with the insertion of a GS linker between IFN-α2b and the IgG1 Fc fragment. The amplified gene was digested with the restriction enzymes BamH I and EcoR I and was inserted into the same digested pPIC9 vector. This recombinant expression vector is denoted as pIFN-α/Fc-SC.
The primer pairs α-Fw and MD-Rv and MD-Fw and Fc-Rv were used to amplify two parts of the fused coding sequences of the IFN-α/Fc-MD homodimer on the template plasmid pIFN-α/Fc-SC. The PCR procedure was as follows: 94 °C for 5 min; 32 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 60 s; and 72 °C for 10 min. The primer pair α-Fw and Fc-Rv was used to splice the two amplified parts to obtain the complete fused IFN-α2b and the IgG1 gene. The PCR procedure was as follows: 94 °C for 5 min; 32 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 120 s; and 72 °C for 10 min. This newly amplified gene encoding a homo-dimerized IFN-α2b and the IgG1 Fc fragment-fused protein also contained a mutated glycosylation site on the IgG1 Fc fragment with the insertion of a protein linker between IFN-α2b and the IgG1 Fc fragment. This newly amplified gene was digested with the restriction enzymes BamH I and EcoR I and was inserted into the same digested pPIC9 vector. This recombinant expression vector was denoted as pIFN-α/Fc-MD.
Screening for expression
The recombinant expression vectors pIFN-α/Fc-WT, pIFN-α/Fc-MD and pIFN-α/Fc-SC were linearized by the restriction enzyme Sal I. The linearized vectors were transformed into P. pastoris strain GS115. The positive transformants were screened by MD plates and examined by Western blot using anti-human IgG-HRP conjugates according to a previous report .
Fermentation and purification
The fed-batch fermentation process was performed in a 14 L fermentor (NBS BioFlo 115) with 6 L BMGY media containing 4% glycerol according to previous reports with necessary modifications . Specifically, the culture temperature was maintained at 25 °C, and the pH was controlled at 6.0 with ammonium hydroxide. Moreover, after induction by 100% methanol supplemented with 12 mL/L of PTM1 solution, the DO was set to about 30% air saturation and fermentation was allowed to proceed for less than 18 h to reduce degradation of the targeted products. At the end of fermentation, the pH of the fermentation broth was adjusted to 8.0 by 5 M NaOH and then centrifuged for 20 min at 10,000 g. After centrifugation, the supernatant was filtered through 0.45-μm hollow fiber membranes using the FlexStand benchtop system. A HiTrap MabSelect affinity chromatography column was used to capture the recombinant IFN-α/Fc-fused proteins from the clarified supernatants. The captured proteins were further purified with HiPrep Sephacryl S-200 HR size-exclusion chromatography and stored at −80 °C.
Characterization of the fusion proteins
SDS-PAGE under reduced and non-reduced conditions was performed to analyze the purified proteins. Approximately 10 μg of each IFN-α/Fc fusion protein was loaded and separated by 10% SDS-PAGE; the gel was stained with Coomassie brilliant blue. Western blotting was also performed using the anti-IFN-α monoclonal antibody or anti-human IgG-HRP conjugates. The protein samples were separated by 10% SDS-PAGE under reduced conditions and then transferred to a PVDF membrane for 30 min at 18 V. The membrane was blocked with 5% skim milk for 1 h and then incubated with the indicated antibodies. The membrane was washed three times with TBST and then incubated with anti-mouse HRP-conjugated secondary antibodies. The protein was detected using a chemiluminescence detection kit after the membrane was washed three times with TBST. A Periodic acid-Schiff (PAS) staining kit (Catalog No. DG0005, Beijing Leagene Biotechnology) was used according to the manufacturer’s instructions to characterize the glycosylated modification of the fusion proteins. The recombinant proteins were also analyzed by liquid chromatography–mass spectrometry (LC–MS), which was performed as previously described .
Antiviral activity assay
WISH or MDBK cells were diluted with DMEM containing 10% fetal bovine serum (FBS) to 2.5 × 105–3.5 × 105 cells/mL. Each well of a 96-well plate was seeded with 100 μL of these diluted cells. Approximately 6 h later, the medium was replaced with DMEM containing 7% FBS and serially diluted samples. After 24 h, the medium was replaced with DMEM containing 3% FBS and 100 TCID50 VSV. Another 24 h later, the cell viability was measured using an MTT assay. The activities of different samples were calculated using Origin 8 software. The experiments were run in triplicate.
Daudi cells were diluted with DMEM containing 10% FBS to 5 × 105 cells/mL. Each well of a 96-well plate was seeded with 50 μL of the diluted cells. Then, 50 μL of DMEM containing 10% FBS was added, and the samples were serially diluted. After 72 h, the cell viability was measured using the MTT assay. The EC50 values of different samples were calculated using Origin 8 software. The experiments were run in triplicate.
2′, 5′-oligoadenylate synthetase (OAS) mRNA assay
Human PBMCs were isolated from the peripheral blood of healthy adult volunteers using Ficoll density gradient centrifugation and diluted with RPMI 1640 medium containing 10% FBS to 1.5 × 105–2.0 × 105 cells/mL. Each well of a 6-microwell plate was seeded with 2 mL of the diluted cells. After 12 h of incubation, the indicated samples were added to each well at a final concentration of 10 ng/mL. After 20 h of incubation, the total RNA of the cultured cells was isolated with TRIzol extraction. Real-time quantitative RT-PCR was used to detect the expression of the targeted genes according to a previous report .
Fifteen Sprague–Dawley (SD) rats with weights ranging from 180 to 220 g were randomly and equally divided into five groups. A 20% ethyl carbamate solution at a dose of 5 mL/kg was used for animal anesthesia. After anesthesia, the indicated samples were injected intravenously into corresponding animal groups with a single 30 μg/kg dose. Blood samples of the treated group were collected prior to dosing and at the following times after dosing: 0.2, 8, 24, 48, 72, 96, 120 and 144 h. Heparin sodium was used for anticoagulation, and then the blood samples were centrifuged for plasma harvest. The interferon levels of the blood samples were analyzed using cytometric bead array (CBA) according to the manufacturer’s instructions. The pharmacokinetic parameters were calculated using the software PKsolver with a non-compartmental model .
Difference between groups was analyzed by applying the one-way analysis of variance (ANOVA) with Tukey’s test (P < 0.05 as significant, P < 0.01 as highly significant, P < 0.001 as very highly significant).