Ktaprime plus protein purification system

His_6x-tagged hRNF145 RING protein was produced in the bacterial RIPL strain (Novagen). Bacteria were grown in Luria Broth (LB) (Sigma) at 37°C to an OD₆₀₀ of 0.6 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h. Bacterial pellets were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.6, 0.5 M NaCl, 5 mM imidazole, and 1 mM DTT) supplemented with protease inhibitors and sonicated on ice to disrupt the cells. Debris was removed by centrifugation, and the cleared lysates were loaded onto HisTrap HP columns (GE Healthcare) coupled to an ÄKTAprime Plus protein purification system (GE Healthcare). Bound proteins were eluted with imidazole, buffer exchanged using Hi-Trap desalting columns (GE Healthcare), and collected in elution buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM DTT). Aliquots were immediately frozen in liquid N₂ and stored at −80°C.

The cDNA sequence of the zinc finger cluster at the N-terminal of MaNCP1 was subcloned and ligated into the expression vector pCold-MBP-TEV to construct the fusion recombinant plasmid pCold-MaNCP1-N. The expressed protein MaNCP1-N was purified by ÄKTA prime plus protein purification system (GE Healthcare, Stockholm, Sweden), followed by concentration and de-salting in a 30-kDa cutoff ultrafiltration tube (Millipore Amicon). The promoter region of MaNmrA, containing a putative binding site for MnNCP1 was amplified with primers Probe-F/Probe-R (Table S3) to produce a 110 bp probe which, was labeled with biotin via the EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China) (59 (link)). The Chemiluminescent EMSA kit (Beyotime, Shanghai, China) was used for the EMSA (59 (link)). The unlabeled probe was added in a 100-fold excess.

Dialyzed samples of the expressed α-amylase inhibitor were also used for purification via size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 75 pg (GE Healthcare) 120 mL column. As such, 15 mL of extract was completely dried under reduced pressure and resuspended in 1 mL of equilibration buffer (PBS 1X, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiotreitol, pH 7.4). Afterward, the column was washed with 120 mL of distilled water at a flow rate of 1 min/mL and then equilibrated with 240 mL of equilibration buffer at the same flow rate. The protein solution was loaded on the column, and 180 mL of equilibration buffer was injected at a continuous flow rate of 1 min/mL for elution; fractions were collected every 2 min. Chromatography was performed using an ÄKTAprime plus protein purification system (GE Healthcare), and chromatogram peaks at 280 nm were generated and analysed by UNICORN 6.4 software (GE Healthcare). Ninety fractions (2 mL each) were collected, and 15 μL of each fraction of the different peaks were separated by 15% (m/v) SDS-PAGE for silver staining according to the methods of Switzer et al. [54 (link)]. Fractions corresponding to the α-AIC3 peak were combined, lyophilized, resuspended in ultrapure water and quantified. Aliquots of 20 μg of proteins were separated by electrophoresis using 15% (m/v) SDS-PAGE.