Figure 2.

Expression of recombinant NfCPI and structural analysis. (A) The NfCPI was purified using Ni–NTA affinity chromatography and analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1, non-induced E. coli lysate; lane 2, isopropyl-1-thio-β-d-galactopyranoside (IPTG)-induced E. coli lysate; lane 3, Ni–NTA affinity purified NfCPI. (B) The NfCPI was mixed with different concentrations of SDS (0–10%, v/v) and β-ME with or without heating and analyzed via SDS-PAGE. (C) Gel filtration chromatography analysis. The NfCPI was loaded to Superdex 200 HR 10/30 column and fractions (0.5 mL) were collected. The collected fractions were analyzed by SDS-PAGE and their inhibitory activities against NfCB and NfCBL were assayed. The fractions expressing strong inhibitory activity (fractions 26 to 28) are represented as red bars and confirmed by SDS-PAGE. The K_av value of NfCPI (red dot) was calculated by comparing with those of size markers (black dots).