Glutathione sepharose resin

Both GST fusion with full length and truncated H-rev107 and POR were isolated and purified from E. coli over-expressing the respective proteins. We expressed the inserted gene via induction of T7 polymerase with 1 mM isopropyl β-D-1-thiogalactopyranoside in BL21(DE3) E. coli after cells reached mid-log phase. Purification of GST fusion proteins was facilitated by glutathione-Sepharose resin (Amersham Biosciences). Purified proteins were confirmed by western blot analysis using antibodies against polyhistidine (Sigma) (data not shown). For in vitro pull-down assay, H-rev107-myc or POR-Flag were in vitro transcribed and translated in TNT^® Quick Coupled Transcription/Translation Systems (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instruction. In vitro transcribed and translated H-rev107-MYC were incubated with 10 μg GST-POR deletions, whereas in vitro transcribed and translated POR-FLAG interacted with GST-H-rev107 deletion variants at 4°C for 2 h with in vitro interaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% (v/v) glycerol, 1% Triton X-100, and a mix of protease inhibitors), after washing three times with interaction buffer and twice with PBS, bound proteins were eluted with 1×SDS sample buffer, separated by denaturing SDS-PAGE, and analyzed with anti-FLAG or anti-MYC antibodies.

Proteins were expressed in BL21 cells grown at 37 °C to an OD600 = 0.6, then induced with 0.5 mM IPTG and incubated for a further 9 hours at 20 °C as previously described^{57 (link)}. The GST-fusion proteins were purified on Glutathione Sepharose resin (Amersham Bioscience). The resin was washed with buffer A (50 mM Tris pH7.5, 1 mM EGTA, 1 mM EDTA and 2 mM DTT) followed by buffer B (50 mM Tris pH7.5, 200 mM NaCl and 2 mM DTT). Bound protein was eluted with elution buffer (50 mM Tris pH8.5, 10 mM glutathione). Eluted protein was filtered, concentrated and frozen in aliquots before use.

Affinity pull-down assay was performed in Poly-Prep chromatography columns (Bio-Rad, USA) with Ni-Nitrilotriacetic acid (Ni-NTA) resin (Invitrogen, USA) and glutathione sepharose resin (Amersham) respectively. The recombinant His-PknI was over-expressed and purified as mentioned (Gopalaswamy et al., 2004 (link)). The nickel affinity pull down was performed by incubating the cytoplasmic crude lysate containing recombinant His-PknI protein with Ni-NTA resin for 2 h at 4°C under gentle agitation. The columns were washed with buffer A containing 40 mM imidazole and incubated with the cytoplasmic crude lysate containing GST-Rv2159c recombinant protein for 4 h at 4°C under gentle agitation. Unbound proteins were removed by washing the column with 10 column volumes of buffer A. The interacting proteins were eluted with buffer A containing 200 mM imidazole. In the same way, glutathione affinity pull down assay was also performed by incubating the crude lysates containing recombinant GST-Rv2159c and His-PknI proteins with glutathione sepharose resin and eluted using buffer A containing 20 mM reduced glutathione. Eluted proteins were separated by SDS-PAGE and subjected to far-western blotting (FWB).