Hiload 16 60 superdex 200 prep grade

12 h after gene expression induction the cells were harvested by centrifugation and resuspended in 50 mM Na-Phosphate buffer (pH 6.0) containing 1,5 M ammonium sulfate for the following purification step. The cells were disrupted by french pressure cell, and centrifuged at 20,000×g for 30 min at 4°C. The resulting supernatant was applied to a preequilibrated column of phenyl sepharose (HiLoad 26/10 Phenyl Sepharose HP; GE Healthcare, Munich) for hydrophobic interaction chromatography (HIC) and eluted from the column with a stepwise decreasing gradient of ammonium sulfate. The recombinant protein-containing fractions were pooled, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), concentrated and further purified by size exclusion chromatography (HiLoad 16/60 Superdex 200 prep grade; GE Healthcare, Munich). As a final purification step the pooled fractions resulting from size exclusion chromatography were applied to immobilized metal ion affinity chromatography using Ni²⁺-NTA (“Qiaexpress Kit, Qiagen”). The enzyme was eluted from the column with 50 mM phosphate buffer (pH 6.0) containing 250 mM imidazole. Protein concentration was determined by the method of Bradford using bovine serum albumin (BSA) as a standard [28] . The purified protein was dialyzed against 50 mM phosphate buffer (pH 6.0) and the purity was analyzed by SDS-PAGE.

Fractions containing the target protein from the last step were concentrated by an Amicon Ultra-15 ml concentrator (10 kDa Centrifugal Filter Unit, Merck-Millipore) to 5 ml and then loaded onto a Superdex-200 column (HiLoad 16/60 Superdex 200 prep grade, GE) that was preequilibrated with equilibration buffer (20 mM PB, 0.15 M NaCl, 10% glycerol pH 7.2). The fractions collected in this step were also analysed by SDS-PAGE. N-glycosylation was verified by the PNGase F enzyme digestion method [15 (link)].

The collected media with the AXL54-x-CD3 protein was purified by HIS GraviTrap™ Kit (GE Healthcare). The column was packed with Ni Sepharose Fast Flow resin. The eluted protein was concentrated to a 5 mL volume using an Amicon Ultra-15 Centrifugal Filter Unit 3 KDA MWCO (Millipore, Burlington, MA, USA). The concentrated protein was purified by SEC on an ÄKTAprime FPLC (Amersham Biosciences, Amersham, Buckinghamshire, UK). The column was a HiLoad 16/60 Superdex-200 prep-grade (GE Healthcare). The column was calibrated using the Gel Filtration Calibration Kit LWM (GE Healthcare). The column was equilibrated in 400 mL Gel Filtration Buffer (1× PBS, 10 mM Sodium Phosphate, pH 7.4 and 150 mM NaCl) at 0.5–1.0 mL/min overnight. The sample loop was washed out with 25 mL Gel Filtration buffer and 5 mL sample was loaded into the loop. The column ran at a flow rate of 0.5 mL/min, 200 mL elution volume, 4 mL fractions. A total of 10 µL of protein was run on SDS-PAGE gels and stained with SimplyBlue SafeStain (ThermoFisher Scientific) to identify the fractions with the protein. The peak fractions were pooled and concentrated to 10 mL with Amicon MWCO 3 kDa. The purified protein was dialyzed overnight in 4 L Storage Buffer (50 mM sodium acetate buffer, pH 4.5, 500 mM NaCl) at 4 °C using SnakeSkin Dialysis Tubing (ThermoFisher Scientific). Protein was stored at −80 °C.

AE and neoPTE genes were cloned into pET32-trx plasmid without Strep-tag using FastDigest NcoI and HindIII as described above, transformed into E. coli BL21 (DE3), and grown for 72 hr at 30°C in TB medium containing 100 μg/ml ampicillin and 500 μM ZnCl₂. Cells were harvested by centrifugation at 3320×g and 4°C for 10 min, resuspended in 20 mM Tris-HCl pH 8 containing 100 μM ZnCl₂ and lysed by sonication (OMNI Sonic Ruptor 400, Thermo Scientific, Waltham, MA, United States, 3× 30 s on/60 s off, amplitude 40%). Cell debris was removed by centrifugation at 30,000×g and 4°C for 45 min and lysate filtered through 45 μm filters (Millipore). The lysate was loaded onto two HiPrep Q FF columns (GE Healthcare, Wauwatosa, WI, United States) in series. PTE elutes in the flow through as well as the early wash fractions. Active fractions were pooled and passed through a 45 μm filter. The sample was concentrated over a Millipore spin column (MWCO 30,000) and purified by gel filtration (HiLoad 16/60 Superdex 200 prep grade, GE Healthcare, Wauwatosa, WI, United States). Protein was concentrated to 12 mg/ml and stored at 4°C.

Human Apo D (hApo D) was purified from BCF samples provide by the Pathology Unit of the Hospital Universitario Central de Asturias (HUCA). First, a cell fractionation was performed by differential centrifugation. Then, Amicon^® Ultra-15, 100 kDa, centrifugal filter units (Z740211, Sigma-Aldrich, St. Louis, MO, USA) were used. The solution containing the protein was flow through two consecutively ion-exchange chromatographic columns (HiTrap^® Q Fast Flow, GE Healthcare, Chicago, IL, USA) with 25 mM Tris pH 8.0, followed by a size-exclusion chromatography (HiLoad^® 16/60 Superdex^® 200 prep grade, GE Healthcare, Chicago, IL, USA ) in 50 mM Tris pH 8.0, 75 mM NaCl. Elution fractions with the protein of interest can be further concentrated using an appropriate 30 kDa cut-off Amicon^® centrifuge filter (Z717185, Sigma-Aldrich, St. Louis, MO, USA). The presence of hApo D in these fractions was checked by Western blot, the amount of this apolipoprotein (concentration in the fraction) was quantified and its functionality was tested.

The genes for p34ct full-length (1–429) and p34ct 1–277 were cloned into the pBADM-11 vector (EMBL) using ligase independent SLIC cloning [21] . Protein expression was carried out in E. coli BL21-CodonPlus (DE3) RIL cells grown in Lennox LB medium. After induction with 0.05% L−(+)−arabinose at an OD₆₀₀ of 0.8 the cells were further grown for 18 – 20 hours at 15 °C.
The full-length protein as well as the shortened variant were purified using Ni-metal affinity chromatography (Ni-TED, Macherey-Nagel) followed by size exclusion chromatography (HiLoad 16/60 Superdex 200 prep grade, GE Healthcare) in either 20 mM Tris-HCl pH 8.0 or 20 mM CHES-NaOH pH 9.5, 150 mM KCl and 1 mM TCEP. The samples were concentrated via Vivaspin filtration units (Sartorius) and the concentration was spectrophotometrically determined based on their theoretical molar absorption coefficients of 28,420 M⁻¹ cm⁻¹ (p34ct) and 26,930 M⁻¹ cm⁻¹ (p34ct 1–277), respectively. After flash freezing in liquid nitrogen, the protein was stored at −80 °C.