Murine CD40L DNA was synthesized using the Pichia pastoris
preferred codons [10
]. Ten primers () were designed to cover the full length of the murine CD40L soluble form (149 aa). There was about a 21 base overlap between any of the neighboring primers. Ten pmol of the first and the last primer, and two pmol for the rest of the primers were used. The PCR program was conducted at 95° C for 5 min, 25 cycles of 95° C for 30 sec, 55° C for 30 sec, 72° C for 1 min and an extension at 72° C for 10 min. The PCR products were analyzed with 1% agarose gel electrophoresis, and the band with the correct size was cut out and extracted with QIAquick Gel Extraction Kit. The synthesized and recovered DNA was digested using Xho
I and Eco
RI and cloned into pwPICZalpha [11
] and designated as CD40L monomer construct after sequencing confirmation. To facilitate the downstream purification, six histidines (6xHis tag) were added at the C-terminus by PCR amplification using primers 40L1 and 40L10His. The PCR product was digested with Xho
I + Eco
RI and cloned back into pwPICZalpha and sequenced to generate CD40L monomer as final construct carrying 6xHis tag in its C-terminus.
PCR primers to synthesize the codon-optimized soluble single chain CD40L monomer, dimer and trimer as well as the his tag in their C-terminus
In order to generate the soluble single chain murine CD40L dimer and trimer expression constructs () the first CD40L was PCR amplified using sense primer CD40L1 carrying XhoI site, and antisense primer CD40L10B carrying BamHI and EcoRI sites. The PCR product was digested using XhoI + EcoRI and cloned into pwPICZalpha for sequencing confirmation. Then it was further digested using BamHI + EcoRI, resulting in a vector construct which contained the first CD40L. The second CD40L was amplified using sense primer CD40L1B carrying XhoI and BglII sites, and antisense primer CD40L10B carrying BamHI and EcoRI sites. The PCR product was digested using XhoI + EcoRI and cloned into pwPICZalpha for sequencing confirmation. BglII + EcoRI were used to cut out the second CD40L insert (BglII-CD40L-EcoRI) which were ligated into the BamHI/EcoRI digested vector pwPICZalpha-CD40L resulting in the construct containing two CD40L (XhoI-CD40L-BamHI/BglII-CD40L-BamHI-EcoRI). The third CD40L insert was prepared by PCR amplification using sense primer 40L1C carrying XhoI and BamHI sites, and antisense primer 40L10His carrying stop codon and EcoRI site. The PCR product was digested using XhoI + EcoRI and cloned into pwPICZalpha for sequencing confirmation. BamHI + EcoRI were used to cut out the third CD40L insert which were cloned into the pwPICZalpha-CD40L dimer vector to generate the CD40L trimer construct (XhoI-CD40L-BamHI/BglII-CD40L-BamHI/BamHI-CD40L-stop codon-EcoRI). The third CD40L insert was also cloned into pwPICZalpha-CD40L plasmid to generate the single chain CD40L dimer construct (XhoI-CD40L-BamHI/BamHI-CD40L-stop codon-EcoRI). The CD40L was tandem linked together by a (G4S)3 linker (Gly-Gly-Gly-Ser) in the dimer and trimer format.
Schematic description of the soluble single chain murine CD40L monomer, dimer and trimer.
Protein expression in Pichia pastoris
5–10 μg of the above constructed plasmid DNA was linearized by SacI digestion for 3h at 37° C, treated with Qiagen PCR purification kit, and transformed into Pichia pastoris strain X33 using the Gene Pulser MXcell Electroporation system (Bio-Rad). Cells were spread on YPD agar plates (1% BactoTM yeast extract, 2% BactoTM peptone, 1.5% BactoTM 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 small tubes containing 5 mL YPD (1% BactoTM yeast extract, 2% BactoTM peptone, 2% dextrose) at 30° C at 250 rpm for 24h as growth phase I, then in YPG (1% BactoTM yeast extract, 2% BactoTM peptone, 1% glycerol) at 30° C at 250 rpm for another 24h as growth phase II. The cultures were induced in 2 mL BMMYC (1% BactoTM yeast extract, 2% BactoTM peptone, 100 mM potassium phosphate, pH 7.0, 1.34% yeast nitrogen base without amino acids (MP), 4 x 10−5 % biotin, 0.5% methanol and 1% BactoTM casamino acids) for 48h at 25° C at 225 rpm. 0.5% methanol was added in 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 supernatant was clarified by centrifugation at 3000 rpm at 4°C for 10 minutes prior to protein purification.
Ni-SepharoseTM 6 Fast Flow was packed in a 5 cm x 20 cm XK50 column (GE healthcare Cat#18-1000-71) 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 filtered through crepe fluted filter paper (VWR) and 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 (5 CV). The bound proteins were eluted with 20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 500 mM imidazole into eight fractions. The purification fractions were analyzed using 12% NuPAGE® Bis-Tris gel and stained with GelCode Blue stain reagent (Thermo Scientific). The fractions containing the protein of interest were pooled and dialyzed using a 3.5 kDa cut off Spectra/Por® membrane tubing (Spectrumlabs) against 20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 5% glycerol at 4° C. The dialysis buffer was replaced once.
Strong cation exchange resin Poros® 50 HS (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 above dialyzed sample was loaded onto the column followed by washing 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 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 a nitrocellulose membrane filter paper using 1x NuPAGE® transfer buffer (Invitrogen). The membrane was thereafter blocked in 2% 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, 0.2% Tween 20 at room temperature with shaking. The murine CD40L monomer, dimer and trimer were detected with mouse anti-His monoclonal antibody (1:500) (Invitrogen) and rat anti-mouse IgG-HRP (1:1000) (Invitrogen) in 2% 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.
ELISA analysis for binding to anti-murine CD40L monoclonal antibody MR1
CD40L variants and BSA were coated to wells of a 96-well polystyrene microplate (Costar 3590) at a concentration of 34 nM in 100 uL of 0.2M sodium carbonate-bicarbonate coating buffer at pH 9.4 (Pierce) by incubating at 4 degrees C overnight. As a positive control, anti-hamster IgG was coated to wells at a concentration of 17 nM (i.e., half the concentration of CD40L since the antibody is bivalent). Wells were then blocked with 300 μL of SuperBlock Protein in 2.5 mM Tris, 0.15 M NaCl, pH 7.4 (Pierce) for at least 2 hrs at room temperature. Wells were then washed 3–6 times with a 0.1% Tween 20 solution at pH 7.4. Biotinylated hamster anti-mouse CD40L monoclonal antibody clone MR1 (National Cell Culture Center, Minneapolis, MN) was added in duplicate to appropriate wells at a concentration of 1, 0.5, 0.25, 0.125, 0.063, 0.031, or 0.016 μg/mL in 100 μL blocking solution with 0.1% Tween 20 and incubated at room temperature for 1–2 hours. After washing, 100 μL of streptavidin-HRP at a concentration of 0.2 μg/mL were added to the wells. The plate was incubated for 30 minutes at room temperature before washing and adding 100 μL of TMB (tetramethylbenzidine/chromagen) substrate. The plates were covered and incubated in the dark at room temperature for 10–30 minutes before stopping the reaction with 100 μL of 1 N HCl. Binding was determined by measuring absorbance at 450 nm within 30 minutes of stopping the reaction.