Aminolink plus coupling resin

Peptide-HLA-A*02:01 complexes were purified according to a previous report^{39 (link)} with minor modifications. A total of 3.6 L of collected culture supernatant (1.2 × 10⁹ cells) was pre-cleared with inactivated AminoLink Plus Coupling Resin (Thermo Fisher Scientific) to remove nonspecific proteins, followed by immunoprecipitation with anti-CII mouse monoclonal antibody (6G4)-immobilised AminoLink Plus Coupling Resin (Thermo Fisher Scientific). After a series of wash steps, the interacted epitope peptides were dissociated from HLA molecules using 10% acetic acid at 90 °C for 5 min and then passed through a 10 kDa molecular mass cutoff filter (Millipore Corp, Bedford, MA). The isolated peptides were fractionated using a MonoSpin SCX spin column (GL Sciences Inc., Tokyo, Japan) according the manufacturer’s instructions. The eluate from 350 mM NaCl was desalted using a MonoSpin C18 spin column (GL Sciences Inc.) according to the manufacturer’s instructions and dried using a savant SPD2010 SpeedVac system (Thermo Fisher Scientific).

The purification of the specific antibody was performed using AminoLink Plus Coupling Resin according to the manufacturers' instructions (MicroLink, Thermo Scientific, USA). We purchased recombinant human Tyro3 protein (Abnova, Taiwan) and coupled the protein to the resin. Then, we used the purified total IgG from SLE patients to form the resin-bound complex and incubated them with gentle end-over-end mixing. The eluted antibody was neutralized with 1 M Tris (pH = 9.0). After sterile filtration, the autoantibody was stored as small aliquots at -80°C.

A region of 597 bp (corresponding to the 199 N-terminal amino acids) from atrR were PCR amplified and cloned in frame as BamHI/SalI fragments downstream of the 6×-His tag in pET28a+ (EMD Millipore, Inc.) to form pGT3 and transformed into the bacterial strain Escherichia coli BL21(DE3). Two liters of transformed bacteria was grown to log phase and induced with isopropyl-β-d-thiogalactopyranoside (IPTG) for 90 min. Cell lysates were prepared using a French press. Protein purification was accomplished using Talon metal affinity resin (TaKaRa Bio USA, Inc.) as described by the manufacturer. Protein fractions were analyzed by staining them with Coomassie blue and by Western blotting using His-specific antibodies. The purified proteins were then lyophilized and sent to Pacific Immunology (Ramona, CA) for injection into rabbits to generate polyclonal antibodies against AtrR. Antiserum generated from these rabbits was received and tested for immunoreactivity against A. fumigatus cell lysates. The antiserum was then purified using an AminoLink Plus coupling resin (Thermo Scientific, Inc.) according to the manufacturer’s instructions, and the affinity-purified antiserum was used to detect the AtrR protein from A. fumigatus cell lysates.