Human fxiia

Inhibitory activities of AaTI and Inf4 on human FXIIa (Merck) were assessed via chromogenic assays using the Chromogenix S-2302 chromogenic substrate (Diapharma; West Chester, OH). For IC₅₀ determination, FXIIa was pre-incubated with different concentrations of inhibitors. The generation of p-nitroaniline (pNA) was determined by measuring the absorbance at 405 nm using a absorbance plate reader (Tecan; Männedorf, Switzerland). The reaction mixture consisted of 40 µl FXIIa (10 nM), 40 µl inhibitor, and 40 µl chromogenic substrate (1 mM). All reaction components were prepared in 50 mM Tris pH 7.4 containing 100 mM NaCl.

Bacterial codon optimized genes encoding for AaTI and Inf4 were purchased from Bio Basic Asia Pacific Pte Ltd (Singapore) and subcloned into a modified version of the pET32a (Merck; Kenilworth, NJ) prokaryotic expression vector. Protein expression was carried out using IPTG (isopropyl β-d-1-thiogalactopyranoside) induction in SHuffle T7 competent Escherichia coli cells (New England Biolabs; Ipswich, MA). His-tagged proteins were purified using the cOmplete His-tag purification resin (Merck) and the eluate was further purified using size-exclusion chromatography using Hiload 16/600 Superdex 75 pg column (16 × 600 mm) (GE Healthcare; Chicago, IL). The His-tag remains intact in all the recombinant wild-type and chimeric proteins used in this study. Purified proteins were tested for inhibitory activities against human FXIIa (Merck). All wild-type, mutant and chimeric proteins used in this work were dissolved in a 50 mM Tris-HCl, pH 7.4 buffer, with 200 mM NaCl.