Tev cleavable c terminal mbp fusion tag

The Apn2^FL (full length) and Apn2^Cat (residues 1–407) were PCR amplified and subcloned into the pET MBP His6 LIC cloning vector (2Cc-T), containing a TEV-cleavable C-terminal MBP fusion tag (Addgene). Apn2^{Cat(D222N/E59Q)} was cloned by site-directed mutagenesis of the Apn2^Cat construct. Apn2^FL mutants were cloned by site-directed mutagenesis of the Apn2^FL construct. The wedge loop deletion construct was made by replacing Apn2 residues W312 through Y323 with two glycine residues. BL21-AI cells (Life Technologies) were transformed with the constructs for protein expression overnight at 15°C in Terrific Broth. Induction of expression was carried out by the primary addition of 0.1% (w/v) final concentration L-Arabinose (GoldBio). Following Amylose affinity chromatography, MBP fusion proteins were diluted in ddH20 and loaded onto 5-mL ion exchange columns for gradient elution (GE Healthcare; HiTrap Heparin). For crystallography, proteins were subjected to overnight TEV protease cleavage for MBP tag removal prior to ion-exchange chromatography. Purest fractions were pooled and purified by Superdex 200 (GE Healthcare) gel filtration for final polishing. Final purity was assessed by SDS-PAGE and fractions pooled and concentrated for subsequent experiments. The yeast PCNA protein was a kind gift from Andrea Kaminski.