Recombinant Protein Purification and Activation

InpA was produced as a recombinant protein in Escherichia coli and purified as previously described [12 (link), 13 (link)] by affinity chromatography on Fast Flow Ni-NTA (Ni²⁺-nitrilotriacetate)–Sepharose (Qiagen) followed by anion exchange chromatography (MonoQ, GE Healthcare). The active concentration of InpA was determined by active-site titration using an appropriate dilution of a standardized 1 mM aqueous solution of the protease inhibitor N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64) (Sigma-Aldrich; product E3132) needed for total inactivation of the proteinase. Residual enzyme activity was determined by measurement of fluorescence (λ_ex = 380 nm and λ_em = 460 nm) of AMC (7-amino-4-methylcoumarin) released from t-butoxycarbonyl-Val-Leu-Lys-AMC as described previously [13 (link)]. Immediately before use, InpA was activated by incubation for 15 min with 3 mM DTT in 0.1 M Tris–HCl, 0.14 M NaCl, pH 7.5 [50 ]. When used for protein breakdown assays under non-reducing conditions, the above buffer was exchanged for that without reducing agent by ultrafiltration using 10 kDa cut-off Microcons (Amicon).