Purification and characterization of active recombinant human napsin A

摘要: Recombinant human napsin A expressed in embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of propeptide as affinity ligand. N-Terminal amino-acid sequencing the enzyme identified mature form A. Treatment with endoglycosidases F and H resulted decrease its molecular mass from 39 kDa ≈ 37 kDa, confirming that is glycosylated. The kinetic properties were analyzed two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ–Lucifer yellow (DS1) K(Dabsyl)-TSVLMAAPQ–Lucifer (DS3). Km values obtained 1.7 µm 6.2 µm, respectively. substrate-specificity study A-targeted peptide library confirmed preference for hydrophobic residues at positions P1 P1′. Adjacent positions, P2–P4 P2′–P4′, appeared less restricted distribution amino acids. pH optimum between 4.0 5.5 room temperature determined. fully active more than 10 h pH 5.0 6.0, while half-life 4 h determined pH 7.0 37 °C.