Pd 10 column

BSA was modified with 2-(4-isothiocyanatobenzyl)-cyclohexyl-diethylenetriaminepenta-acetic acid (SCN-CHX-A”-DTPA, Macrocyclics) as previously described [15 (link)]. In brief, BSA and non-specific antibodies were incubated with 20 equivalents (mol/ mol) of CHX-A”-DTPA in carbonate buffer (0.05 M, pH 8.7) overnight at room temperature and then purified by HPLC on a Sephadex G200 gel-filtration column (Amersham Biosciences, Saclay, France). The mean chelate number per BSA molecule was 2, as assessed with 4 equivalents of citrate-acetate (0.02–0.15 M, pH 5.5) buffered ¹¹¹In solution. For labelling with ²¹³Bi, the BSA-CHX-A”-DTPA was incubated with ²¹³Bi eluted from a ²²⁵Ac/²¹³Bi generator (Institute for Transuranium Elements, Karlsruhe, Germany) for 10 min in 0.8 M ammonium acetate (pH 5.3). The resulting ²¹³Bi-labelled BSA was separated from unbound ²¹³Bi by size-exclusion chromatography using a PD-10 column (GE Healthcare) [16 (link)]. Radiochemical purity, checked by ITLC-SG using 10% TCA as solvent [17 (link)], was greater than 95%.
BSA was labelled with ¹²⁵I (Perkin Elmer, Courtaboeuf France) using the iodogen method as described previously [18 (link)]. The ¹²⁵I-labelled BSA (¹²⁵I- BSA) was purified on a PD10 column (GE Healthcare). Radiolabelling efficiency, estimated by ITLC, was above 95%.

The protein was expressed as His-tagged recombinant proteins in DH10B E. coli cells. The culture was incubated at 37 °C for 4 h when OD achieved 0.6–0.8. Then, L-arabinose was added to a final concentration at 0.02%. Then, the culture was transferred to 30 °C for overnight incubation for a maximum of 18 h. Bacterial culture was harvested at 10,000 rpm at 4 °C for 10 min. Cell pellet was resuspended in Tris-buffered saline (TBS, 150 mM NaCl, 20 mM Tris, pH 7.5) and lysed by using sonication. The His-tagged protein from the collected supernatant was purified by affinity chromatography using Ni-NTA beads. Protein-bound beads were washed with wash buffer (20 mM imidazole, 50 mM Tris pH 7.5, 300 mM NaCl) and eluted with an elution buffer (50 mM NaH₂PO₄.H₂O, 300 mM NaCl, 500 mM Imidazole). The eluted protein fractions were buffer exchanged by using a PD-10 Columns (GE Healthcare Life Sciences, Chicago, IL, USA) desalting column and then concentrated by using 10 kDa cut-off centrifuge filter columns (Amicon, Merck Millipore, Burlington, MA, USA) and stored in 1× TBS (pH 7.5).

The genes xyn65 and xyn80 without the signal peptide were cloned from the genomic DNA of strain HB14^T by PCR amplification and inserted into the pET22b vector (Novagen, Germany). The primers used are shown in Supplementary Table S1. The constructed plasmids were transferred into E. coli BL21(DE3; Vazyme, China). The cells were cultivated in the Luria-Bertani (LB) medium containing 0.1 mg/ml ampicillin at 37°C and 180 rpm to OD₆₀₀ ≈ 1.0 and then induced at 18°C and 120 rpm for 18 h with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation (11,500 g for 5 min at 4°C) and lysed in a lysis buffer [50 mM Tris–HCl, 100 mM NaCl, 0.5% (v/v) glycerol, pH 8.0] by a pressure crusher (JNBIO JN-02C). The recombinant proteins were purified by the nickel-nitrilotriacetic acid resin (GE Healthcare, United States) and then desalted by PD-10 columns (GE Healthcare, United States). Protein purity and apparent molecular weight were determined by SDS-PAGE. Protein concentration was determined by the bicinchoninic acid (BCA) method using a BCA protein assay kit (Thermo, United States).

Serum was fractionated by fast-protein liquid chromatography (FPLC) and apolipoprotein A-I-containing fractions were pooled and concentrated using Amicon Ultra-4 centrifugal 10 K filters (Millipore, Merck, Darmstadt, HD, Germany). Pooled fractions were subjected to dynabead-based immunoprecipitation with an anti-apolipoprotein B (apo B) antibody to remove apo B-containing lipoproteins. Apo B lipoprotein-depleted supernatants were subjected to discontinuous density gradient ultracentrifugation to isolate high density lipoproteins (1.063 < d < 1.21 g/mL). After centrifugation, HDL fractions were collected, desalted via PD10 columns (GE Healthcare, Vienna, WI, Austria), and immediately used for experiments.

The P. pastoris wild type strain (X-33), the pPICZB vector, zeocin, Ni-NTA agarose resin were from Invitrogen; PD-10 columns were from GE Healthcare; L-[³H]Glutamine and L-[³H]glutamic acid were from Perkin Elmer; C₁₂E₈ was from TCI Europe; Cholesterol, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), Sephadex G-75, L-glutamine, L-glutamic acid monosodium salt, DEPC, valinomycin, nigericin, pyranine (8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) and all the other reagents were from Sigma-Aldrich.