Desalting column

All genes were amplified by PCR and cloned into the pET.28a vector to produce His6-tag-fused recombinant proteins, or the pGEX-6P-1 vector to produce GST-tag-fused recombinant proteins. All recombinant proteins used in this study were expressed in E. coli BL21-CodonPlus (DE3) induced by 0.3 mM IPTG for 16 h at 20 °C and collected by sedimentation.
To purify His-KIN-3, His-KIN-10, His-TOFU-4, HIS-TOFU-4(S131A S229A) GST-KIN-3 and GST-tagged TOFU-4 fragments, the E. coli cells were resuspended in binding buffer (50 mM Tris-Cl pH 7.9, 500 mM NaCl and 10 mM imidazole), lysed with a high-pressure homogenizer and sedimented at 18000 rpm for 30 min at 4 °C. The supernatant lysates were purified on Ni-NTA agarose beads (for His6-tagged proteins, Qiagen) or Glutathione High Capacity Magnetic Agarose Beads (for GST-tagged proteins, SIGMA). After 2 extensive washing with binding buffer, the proteins were eluted with His6 elution buffer (50 mM Tris-Cl pH 7.9, 500 mM NaCl and 500 mM imidazole, for His6-tagged proteins) or GST elution buffer (50 mM Tris-Cl pH 7.9, 500 mM NaCl and 10 mM GSH, for GST-tagged proteins). All the eluted proteins were concentrated by centrifugal filtrations (Millipore), loaded onto desalting columns (GE Healthcare), then eluted with PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄). The eluted proteins were stored in aliquots at -80 °C.

ApoA-I WT, Milano (R173C) and A164S proteins were produced and purified as previously described^{30 (link),31 (link)}. In short, a bacterial expression system consisting of pEXP-5 plasmid (Novagen) in Escherichia coli strain BL21(DE3) pLysS cells (Invitrogen) was used to produce the apoA-I proteins. His-tagged ApoA-I proteins were purified from bacterial cell lysate by immobilized metal affinity chromatography (His-Trap-Nickel-chelating columns, GE Healthcare) under denaturing conditions (3 M guanidine in phosphate-buffered saline (PBS), pH 7.4). Following binding, an extensive wash with 40 mM imidazole in PBS was performed and proteins were then eluted with 500 mM imidazole in PBS. Imidazole was removed from protein samples by using desalting columns (GE Healthcare) equilibrated with PBS, pH 7.4, and tobacco etch virus (TEV) protease was employed overnight at 4 °C, in the presence of 1 mM DTT, to remove the His-tag from protein samples. At the end of the incubation, a reverse Ni²⁺-column step was employed in order purify the cleaved ApoA-I from TEV protease and the His-tag. The flow-through containing cleaved ApoA-I protein was desalted into McIlvaine Buffer (165 mM Na₂HPO₄, 17.6 mM citrate, pH 7) and stored at 4 °C prior to use. SDS-PAGE and Blue-native gel (Invitrogen) were used for analyses of lipid-free protein and HDL particles.

For further characterization, VHHs were produced in E. coli in culture flasks according to standard procedures (Marks et al., 1991 (link)). His-tagged VHHs were purified from the periplasmic extracts using the ÄKTAxpress using a combination of immobilized Nickel IMAC and desalting columns, according to the manufacturer’s instructions (GE Healthcare Life Sciences).