Heterologous Expression and Purification of 4F2hc-LAT2 Complex

To heterologously overexpress human 4F2hc-LAT2, a previously characterized and reported P. pastoris clone was used^{12 (link),14 (link),37 (link)}. The recombinant protein was produced in P. pastoris as described^{23 (link)}, solubilized with GDN and purified according to published protocols^{37 (link),38 (link)}. The anticalin D11vs was produced at preparative scale via secretion in E. coli KS272 with a C-terminal Strep-tag II using the plasmid pNGAL98-D11vs^{36 (link)}. After periplasmic protein extraction, the recombinant protein was purified using a Strep-Tactin Sepharose column (IBA, Göttingen, Germany) and finally subjected to SEC in PBS (4 mM KH₂PO₄, 160 mM Na₂HPO₄, 115 mM NaCl pH 7.4) on a HiLoad 26/600 Superdex 75 pg column (Cytiva, Freiburg, Germany). The GDN-solubilized 4F2hc-LAT2 heterodimer was mixed with 4 molar equivalents of D11vs and incubated on ice for 1 h followed by concentration to ~ 10 mg/mL with an Amicon 100-kDa molecular weight cut-off device (Merck, Switzerland). This sample was further purified by SEC using a Superose 6 Increase 3.2/300 column (Cytiva, Switzerland) with 20 mM Bis–Tris propane pH 8.0, 150 mM NaCl, 1% (v/v) glycerol, 0.02% (w/v) GDN as running buffer. Individual fractions were analyzed by SDS-PAGE and used for cryo-EM grid preparation.