Baculodirect

To create a standard for comparison with mouse NK4-like fragments, human NK4 (1–449) corresponding to the protein generated from HGF by human chymase [15 (link)] was produced in High Five insect cells using a tag-free baculovirus construct (BaculoDirect, Invitrogen, Carlsbad, CA) and purified by loading onto a heparin-affinity HPLC column (Toso-Haas; Montgomeryville, PA) and eluting with a linear gradient of 0.5 to 2.0 M NaCl in 10 mM bis-Tris (pH 6.1). To determine whether mouse HGF can give rise to a similar fragment, as predicted by the high degree of sequence conservation between mouse and human HGF within the 17-residue inactivation segment [15 (link)], we incubated recombinant intact mouse HGF (R&D Systems, Minneapolis, MN) with a mouse chymase (mast cell protease 4, mMCP-4) purified from ear skin as described [18 (link)]. We also incubated mouse HGF with recombinant mouse neutrophil elastase (R&D Systems) and cathepsin G (produced as described [19 (link)]). Resulting digests were compared on Coomassie Blue-stained, non-reducing SDS-PAGE gels.

A recombinant baculovirus expressing S. pombe 6His-Fbh1 was constructed using BaculoDirect (Invitrogen) and a pENTR4 derivative, pENTR41-6His-Fbh1. Recombinant baculoviruses expressing S. pombe 6His-Skp1, 6His-Pcu1, and 6His-Rbx1 were prepared using the Bac-to-Bac expression system (Invitrogen).

Soluble pHLA‐DR1 was either refolded from recombinant DR1α and DR1β chains produced in the BL21(DE3) strain of E. coli [29], or generated in Spodoptera frugiperda (sf9) insect cells using the BaculoDirect™ expression system (ThermoFisher Scientific) [30] as previously described. pHLA‐DR1 molecules were biotinylated by inclusion of a C‐terminal AviTAG™ biotinylation signal sequence on the DR1α chain that was biotinylated using a BirA biotin‐protein ligase kit (Avidity) [36]. A fusion protein of the four extracellular domains of LAG‐3 and the Fc domain of IgG (LAG‐3:Fc) was produced in CHO cells as previously described [19]. LAG‐3:Fc was stable in a solution of 20 mM sodium citrate, 86 mM sodium chloride, 100 mM l‐arginine, 0.02% Tween‐20, pH 7.4 with citric acid (TBSB buffer) at concentrations of up to 30 mg/mL.