Figure 3
From: Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells
Expression and purification of soluble dimeric rat NK cell C-type lectin-like receptors. (A) Small-scale expression test of pHLsec constructs; transfection was performed in a 24-well plate, and 10 μl samples of culture supernatants were collected 3 days later and resolved by 15% SDS-PAGE under non-reducing conditions, transferred onto nitrocellulose membrane and detected by PentaHis mAb; lanes: M, marker; 1, mock transfected; 2, RCTL I38-T180; 3, Clr-11; 4, NKR-P1BWAG; 5, NKR-P1BSD; 6, RCTL H51-T180; 7, RCTL H51-T170. Full-size images of membranes and their photographs are available as Supplementary Information (Figure S1). (B) Samples of purified proteins were resolved by 4–20% SDS-PAGE under both reducing and non-reducing conditions; lanes: pHLsec_Clr-11, pHLsec-FcHis_Clr-11 cleaved off by a 3C protease, pYD5_NKR-P1BSD (monomeric and dimeric fractions) and pYD5_ NKR-P1BWAG, both cleaved off by TEV protease. (C) Gel filtration profiles of Clr-11, Clr-11-FcHis, and Clr-11 with the FcHis tag cleaved off by a 3C protease. (D) Gel filtration profiles of pYD5 Fc-NKR-P1BSD/WAG fusion proteins and NKR-P1BSD/WAG with the Fc tag cleaved off by TEV protease, resulting in the disulphide dimer of NKR-P1BWAG and a mixture of dimer and monomer species of NKR-P1BSD.
