Glutathione sepharose affinity column

All plasmids used for protein expression were transformed into E. coli strain BL21 (DE3) pLysS cells (Novagen) by standard procedures [74 ]. E. coli cells were cultured at 37°C for about 4 h, followed by addition of 200 μM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma-Aldrich) and shaking at 18°C for 18 h for induction of protein expression. Purification of recombinant proteins was performed as described previously [67 (link), 84 (link)]. Briefly, E. coli cells were harvested, re-suspended in buffer T (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% glycerol, 1 mM PMSF), disrupted by ultrasonication (Model 500 Homogenizer, Fisher Scientific), and centrifuged to obtain the clarified supernatant. For purification of the MBP-tagged αa_HEL protein, supernatants were passed over Ni−NTA agarose columns (Bio-Rad) at least three times to ensure efficient binding to the beads, and the columns were eluted using stepwise increases in imidazole concentration. For recovery of GST-tagged proteins, supernatants recovered after centrifugation were passed over a glutathione-Sepharose affinity column (GE Healthcare), and the GST fusion proteins were eluted with T buffer containing 60 mM L-Glutathione and 2 mM DTT. The eluted recombinant proteins were concentrated with an Amicon Ultra-15 filter unit (Millipore) and used for RNA helicase assays.

GST-tagged MParD2 and GST tag alone (as negative-bait control) were purified as follows: IPTG-induced 500 ml EC18 culture was harvested and lysed by sonication in 20 ml binding Buffer-A (50 mM Tris-Cl pH 7.5, 50 mM NaCl, 5% glycerol and 1 mM EDTA). The lysate was loaded on a glutathione sepharose affinity column (GE Life Sciences) and eluted in Buffer-A containing reduced glutathione. The purified GST-D2 or GST proteins (after dialysis against buffer A) were mixed with partially purified His6X-MParE2 in Buffer A and incubated overnight at 4°C for interaction. The mixture was loaded on a glutathione sepharose column, washed with Buffer-A containing 2 mM reduced glutathione and eluted in Buffer-B (Buffer-A containing 300 mM NaCl and 20 mM reduced glutathione). The eluted fractions were concentrated by Amicon-ULTRA (MWCO 3KDa) filtration. Partially purified His6X-Domain 4 (PA-D4) of the protective antigen of Bacillus anthracis (Manish et al., 2013 (link)) was used as non-specific protein-prey control. Protein samples were resolved using SDS-PAGE and immunoblotting was performed using anti-GST (Santa Cruz biotech) and anti-His (Sigma) monoclonal antibodies.

The recombinant plasmid pGEX-4T-1-scFv-9R, contains cDNA of an anti-EGFR scFv, and an nona-arginine residues (9R) linked to its C-terminus. scFv was designed according to the amino acid sequences of nimotuzumab (a monoclonal antibody against EGFR) with a linker (Gly4Ser3) connecting its VH and VL chains. pGEX-4T-1-S only contains cDNA of scFv fragment and served as a control. scFv-9R and scFv were fused with a 6×His.tag at N-terminus. Single colonies of E. coli BL21 (DE3) carrying the recombinant plasmid pGEX-4T-1-scFv-9R and pGEX-4T-1-S were grown overnight at 37°C in 2×YT medium supplemented with 100 μg/ml ampicillin. The cultures were diluted 100-fold in the same medium, and induced with 0.1 mM isopropyl-1-thio-β-galactopyranoside (IPTG, Wilson, China) at 30°C for 5 h. The fusion proteins were purified from the E. coli cell lysates with a glutathione-sepharose affinity column (GE Healthcare, Uppsala, Sweden) after 30 min of sonication. The GST tag was cleaved by the 25-fold diluted thrombin (Novagen, Darmstadt, Germany) at room temperature for 8 h, and removed from the solution by GST affinity chromatography.