The Pfs25 sequence from 3D7 clone (ACCESSION P13829) Ala 22 to Thr 193, lacking the native signal sequence and GPI-anchor was used to produce recombinant Pfs25 in E. coli, Pichia, and baculovirus. The GPI anchor sequence functions poorly in heterologous eukaryotic system and was not included . Codon optimization for each expression system was performed by DNA2.0 with a C-terminal hexa-histidine tag added to the coding region to facilitate affinity purification.
Pfs25 with and without additional N-terminal periplasmic signal sequence (encoding MKYLLPTAAAGLLLLAAQPAMA of Pectate Lyase B of Erwinia carotovora) and with 5′ NdeI and 3′ XhoI restriction sites was cloned into pET41a (Novagen) via standard cloning procedures. Resulting plasmids denoted as pET41a-peri-Pfs25 (with periplasmic signal peptide) and pET41a-Pfs25 (without signal peptide), were sequenced and verified.
Briefly, two mutations were introduced (N112Q and N187Q) to avoid N-glycosylation . The resulting Pfs25 fragment with 5′ BamHI and 3′ EcoRI restriction sites was cloned into pPIC9K (Thermo Fisher) in frame with alpha-factor secretion signal. The expression plasmid denoted pMBL003-Pfs25 was sequenced and verified. pMBL003-Pfs25 was transformed into Pichia pastoris BICC9682, and cells plated on YNBD(Yeast Nitrogen Base Dextrose) agar plates at 30 °C for 3 days. Approximately 100 clones were screened for the expression of Pfs25 and an additional 2000 clones on G418 plates screened for multi-copy integrants.
Synthetic pfs25 containing N112Q and N187Q mutations as described in Pichia, with an additional N-terminal secretion signal (MKFLVNVALVFMVVYISYIYAD from Honeybee Melittin) was cloned into pFastBac vector (Invitrogen) with BamHI (5′) and EcoRI (3′) sites and the resulting plasmid pFastBac-Pfs25 was sequence verified. The generation of recombinant virus followed Users’ Manual of Bac-to-Bac system (Invitrogen). Briefly, pFastBac-Pfs25 was transformed into E. coli DH10Bac to generate recombinant bacmid and colonies grown at 37 °C for 48 h on LB agar plates containing Tetracycline (10 μg/ml), kanamycin (50 μg/ml), gentamycin (7 μg/ml), IPTG (40 μg/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, 100 μg/ml), according to users’ manual. The bacmids from selected colonies were confirmed by PCR and sequencing, then used to transfect Super Sf9 cells (Oxford Expression Technologies) for the generation of recombinant baculovirus stock (P1), using CellFECTIN II (Invitrogen) following Bac-to-Bac manual. P1 virus (approximately 14 ml) was harvested and stored at 4 °C protected from light. Two-milliliters of the P1 virus was used to amplify P2 baculovirus after infecting fresh super Sf9 cells at 27 °C for approximately 90 h and similarly to produce high titer P3 virus; remaining P1 baculovirus supernatant was stored at −80 °C. A P3 virus volume of 400 ml for further expression was harvested and titered using BacPAK titer kit (Clontech).
Escherichia coli protein expression
Several methods to express soluble Pfs25 in E. coli were used including cell lines BL21 (DE3) and BLR(DE3), co-expression of protein disulfide bond isomerase (DsbC), expression in lower temperature (20 °C), and IPTG concentrations (1 and 0.25 mM). All of which did not yield sufficient quantity of soluble, monomeric Pfs25 for further purification. Briefly, pET41a-Pfs25 (cytoplasmic) and pET41a-peri-Pfs25 (periplasmic) plasmids were transformed in BL21 (DE3) and BLR (DE3), respectively, and clones were selected and verified. Expression induction at 37 and 20 °C was carried out for 3 and 16 h, respectively at an optical density A600 of 0.8. Induced cultures were harvested by centrifugation for 10 min at 4 °C. The cell pellet was collected and lysed using BugBuster Protein Extraction Reagent (Novagen) per the manufacturer’s instructions. Soluble and insoluble fractions were separated via centrifugation.
Pichia protein expression and purification
Approximately 100 clones of pMBL003-Pfs25 in Pichia pastoris were screened for protein expression in 2 ml deep well plates with 2 ml complex medium containing 2 % dextrose for 2 days. Medium was then removed and replaced with 2 ml of complex medium with 1 % methanol for 3 days, with 1 % methanol feed once a day. Samples were collected on the third day post induction. Three high-yield clones were identified by analyses with reducing SDS-PAGE (Pfs25 expression band at 20 kDa), and evaluated with anti-His (Qiagen) or anti-Pfs25 mAb 4B7 [6, 22, 23] (BEI Resources) Western blots as described.
Pichia (G418 screening)
In parallel, clones were screened on YPD (Yeast peptone dextrose) agar in the presence of G418 (0.5, 1 and 2 mg/ml) in 96-well plates—corresponding to potential increased copies of gene of interest and hence, likely increased protein yield. G418 positive clones, including ten clones by 0.5 mg/ml G418, eight by 1 mg/ml G418, and two by 2 mg/ml G418 were subjected to small-scale expression as described earlier, which led to identification of slightly better protein expressers. A selection of ten clones was then evaluated in 100 ml BMGY (Buffered Glycerol complex medium) and induced with BMMY (Buffered Methanol complex Medium) using 1 % methanol feeding per day for 3 days followed by analysis (reducing SDS-PAGE). Two colonies, C6 and H4, produced similar yield and quality of Pfs25, and C6 was arbitrarily selected for 800 ml expression and further purification.
Supernatant from G418 (clone C6) was purified using Ni–NTA resin (Qiagen) using standard protocol. Briefly, 200 ml supernatant was adjusted to 1000 ml with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, applied to column and washed (10 CV, column volume) with 5 mM Imidazole in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl. Proteins were eluted with 300 mM Imidazole in 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, analysed, pooled, and dialyzed into PBS buffer, pH 7.4. The pool was concentrated and further purified by gel filtration (Superdex 75).
Baculovirus expression and purification
Using Super Sf9 cells, three different MOI’s (multiplicity of infection, 1, 3, and 5) were screened at a 30 ml scale as well as uninfected cells (negative control). Post infection (48 and 72 h), 1 ml of culture was collected and centrifuged for 5 min. The supernatant was analysed via reducing and non-reducing SDS-PAGE and Western blot. Based on analysis of Pfs25 post infection of 72 h, no significant difference between the three MOI’s tested was seen and further expression proceeded with MOI = 1 at 100 ml and 72 h.
Supernatant was used for batch binding overnight with ~2 ml of Ni–NTA resin (Qiagen, Cat No: 30410) at 4 °C on table top rocker platform. Two washing steps were used: Wash buffer (1): 1x PBS with 10 mM Imidazole, pH 7.2 (10 CV) and wash buffer (2): 1x PBS with 20 mM Imidazole, pH 7.2 (10 CV). Protein was eluted via a step gradient with elution buffers (1): 1x PBS with 50 mM Imidazole, pH 7.2 and Elution buffer and (2): 1x PBS with 500 mM Imidazole, pH 7.2. Elution fractions were then analysed and those containing Pfs25 were pooled, concentrated, and further purified on Superdex 75 column.
Plant control protein
Pfs25 derived from Nicotiana benthamiana was provided by Fraunhofer CMB (Newark, DE) and utilized as a control antigen. The soluble protein, termed Pfs25MF1E, and encompassing amino acid 23–193 of the Pfs25 sequence, with a theoretical mass of 20,021 daltons is fully described in . The protein was provided at 1.65 mg/ml and in 20 mM Tris buffer containing 70 mM NaCl, pH 8.0 as previously described .
The samples were used with either (1) 4X non-reducing NuPAGE LDS (Lithium dodecyl sulfate) sample buffer (non-reducing and non-boiled) or with reducing agent and heated to 95 °C for 5 min or (2) 4X SDS sample buffer and boiled for 10 min at 99 °C (reducing and boiled). SDS-PAGE gels (4–12 % NuPAGE Bis–Tris or 15 % Tris–glycine) were loaded in a final volume of 20 μl/well and run at 150–200 V for 35–50 min in 1X MES or 1X SDS running buffer.
Following SDS-PAGE, proteins were transferred onto PVDF membrane and blocked in 5 % skim milk (1X Tris buffered saline, TBS) at room temperature for 1 h. Primary antibodies were prepared at 1:5000 dilutions of Penta His antibody (Qiagen Cat No:- 34460), 1:2000 dilution of Anti-Pfs25 mAb 4B7 (BEI), or 1:1000 dilutions of 1G2 (final 2 µg/ml) in 1 % skim milk in 1X TBS buffer containing 0.05 % Tween-20 (TBS-T). Blots were incubated in the primary antibody solution for 1 h at room temperature or overnight at 2–8 °C. Membranes were washed with 1X TBS-T buffer (3X for 10 min) and secondary antibody 1:1000 dilution of goat anti mouse IgG-HRP (Santa Cruz) or 1:4000 goat anti mouse IgG Alkaline Phosphatase (BioRad) in 1 % skim milk (1X TBS-T buffer) was added and incubated at room temperature for 1 h. Membranes were then again washed with 1X TBS-T buffer (3X for 10 min). HRP labeled blots were developed with TMB (3,3,5,5′-Tetramethylbenzidine) substrate. Alkaline Phosphatase labeled blots were developed using Bio-Rad Alkaline Phosphatase Conjugate Substrate Kit.
Kinetic endotoxin assay
Spectramax plus spectrophotometer (kinetic endotoxin assay) was used to quantify endotoxin content of purified Pfs25, with EndoSafe Endotoxin standard (Charles River), EndoSafe Lysate (Charles River), and EndoSafe LAL Reagent Water (Charles River).
Parasite extract preparation
Mature gametocyte cultures were prepared as reported previously . From the stage V gametocytes, zygote- and ookinete-stage parasites were induced in vitro using the method described by Ghosh et al. . The parasite extract containing native Pfs25 was prepared by three times freeze-and-thaw of the P. falciparum NF54 gametocyte/zygote/ookinete parasites. The human serum and red blood cells used for the gametocyte cultures (extract preparation and SMFA) were purchased from Interstate Blood Bank (Memphis, TN).
Size exclusion HPLC (SE-HPLC) analysis
SE-HPLC analysis for purified Pfs25 proteins from Pichia and baculovirus was performed on a BioAssist G3SWxl column (7.8 × 300 mm, TOSOH Biosciences, King of Prussia, PA) with a Shimadzu Prominence UFLC HPLC system at a flow rate of 0.7 ml/min in 0.2 M sodium phosphate pH 6.8. A gel filtration standard (Bio-Rad, Hercules, CA) was used in column calibration.
Samples of Pfs25 were analysed using an Ultimate 3000 UHPLC system (Thermo Scientific) with a 2.6 µm, 2.1 × 150 mm C-18 column (Thermo Scientific) at a flow rate of 0.2 ml/min. C-18 column and auto-sampler temperatures were set at 60 and 5 °C, respectively. The elution of Pfs25 was monitored using absorbance (214 nm). Mobile phase (A) consisted of water with 0.1 % trifluoroacetic acid (TFA) and mobile phase (B) consisted of acetonitrile with 0.1 % TFA with a gradient of: 1 % B (5 min), 1–20 % B (2 min), 20–50 % B (30 min), 50–99 % B (2 min), 99 % B (3 min), 99–1 % B (2 min), and 1 % B (5 min). The Pfs25 samples were also run without column to determine sample recovery (82 ± 2 % for baculovirus protein and 70 ± 3 % recovery for Pichia protein).
Free thiol determination
Free thiol (number of free cysteine residues) in each protein sample was measured using Measure iT free thiol assay kit (Life Technologies, Carlsbad, CA) following manufacturer’s instructions. Samples were diluted in either ultrapure water or 2 M guanidine-HCl and 50 μg of each Pfs25 sample was used for non-denaturing conditions and 25 μg of each Pfs25 sample was used for denaturing conditions. A standard curve (R2 = 0.983) was constructed using known concentrations of reduced glutathione. Fluorescence was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).
Intact mass spectrometry
Intact masses of each sample (in duplicate) were measured using a SYNAPT G2 hybrid quadrupole/ion mobility/TOF mass spectrometer (Waters Corporation, Milford, MA) with the assistance of the Mass Spectrometry and Analytical Proteomics Laboratory at the University of Kansas. The instrument was operated in a sensitivity mode with all lenses optimized on the MH + ion from the control Leucine Enkephalin and sample cone voltage of 40 eV. Argon was admitted to the trap cell operated at 4 eV for maximum transmission. Spectra were acquired at 9091 Hz pusher frequency covering the mass range from 100 to 3000 unified atomic mass unit and accumulating data for two seconds per cycle. Time to mass calibration was made with NaI cluster ions acquired under the same conditions. Mass spectra of [Glu1]-Fibrinopeptide B were acquired in parallel scans and doubly charged ions at m/z 785.8426 were used as a lock mass reference.
Samples were desalted via reversed phase PRP-1 column, 1 cm, 1 mm I.D. (Hamilton, 10 µm particles packed by hand) using a NanoAcquity chromatographic system (Waters Corporation) and solvents A (99.9 % H2O, 0.1 % formic acid) and B (99.9 % acetonitrile, 0.1 % formic acid) over a short gradient from 1 to 70 % B in 4 min with a flow rate of 20 µl/min. MassLynx 4.1 software (Waters Corporation) was used to collect data and deconvolute the protein spectra for molecular weight determination.
Alkylation of Pfs25 and amino acid analysis
Pfs25 protein derived from plant at 1 mg/ml, pH 8.0 was reduced with 20 mM DTT at 60 °C for 30 min and alkylated with 40 mM iodoacetamide at 37 °C for 30 min in the dark; alkylation was quenched with large excess of β-mercaptoethanol. The extent of modified cysteines was analysed by amino acid analysis (AAA) at AI BioTech (Richmond, VA) with pre-column derivation using O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC).
Mouse immunization and SMFA
To produce anti-Pfs25 sera in CD-1 mice for SMFA, 10 µg of Pfs25 was formulated with ISA720 (Montanide) for each of the following groups: Group 1, baculovirus produced Pfs25; Group 2, Pichia produced Pfs25; Group 3, positive control (plant produced Pfs25 ); Group 4, negative control (reduced alkylated plant protein Pfs25) and Group 5, adjuvant only control. The groups of ten mice were injected (intramuscularly) on day 0 and 21. On day 42 sera were collected and individual sera were tested by ELISA. In addition, sera were pooled, IgG affinity purified and tested at 0.75 mg/ml as well as three-fold dilutions (0.250, 0.083 and 0.028 mg/ml) for SMFA activity . Briefly, P. falciparum NF 54 gametocytes and purified IgG from mice immunized with Pfs25 were fed to female Anopheles stephensi mosquitoes. Mosquitoes were then dissected 8 days after the feeding and the number of oocysts counted to measure transmission reduction activity.
Basic methodology of ELISA has been described by Miura et al. . All ELISA plates were coated with plant produced Pfs25  at 100 ng/well. Based on a standard curve generated with a serially diluted ELISA reference standard (a pool of anti-Pfs25 antisera generated against a plant-produced Pfs25 VLP ), a relative antibody level (i.e., ELISA units) of test sample in the same plate was determined.