Glutathione agarose

A synthetic peptide encompassing the C-terminal residues of the MTRAP protein, N-CYFLRKEKTEKVVQEETKEENFEVMFNDDALKGKDNKAMDEEEFWALE-C, was used to generate rabbit polyclonal serum (GenScript). A recombinant protein encompassing valine (residue 95) to glutamic acid (residue 299) was generated by PCR amplification from P. falciparum genomic DNA, cloned by restriction enzyme digestion into a BamHI- and XhoI-digested expression vector (pGEX-4T-1; GE Healthcare Biosciences) and expressed as a recombinant protein in Escherichia coli (strain BL21). The protein was purified over glutathione agarose (Sigma-Aldrich) using conventional methods (Baum et al., 2006b (link)). The purified protein was used to immunise rabbits and generate both polyclonal and a monoclonal antibody (clone 3A3; Walter and Eliza Hall Monoclonal Facility, Bundoora, VIC, Australia).

In vitro GST pull-down assays were used to determine any interactions between the E6 protein and its cellular protein targets (i.e., p53, PDZ1 from MAGI-1, hDLG, E6AP), expressed in E. coli XL1 as GST-fusion proteins. Briefly, E. coli XL1 were grown overnight (16 h), 100-fold diluted in fresh LB with 100 mg/L ampicillin, and incubated at 37 °C until an OD₆₀₀ of 0.6–0.8 was reached. The expression of the recombinant proteins was induced with 1 mM IPTG for 3 h at 37 °C. The proteins were then purified and immobilized on glutathione-agarose (Sigma-Aldrich). All of the immobilized GST-fusion proteins were run on SDS PAGE to evaluate the amount of each protein. Similar amounts (as evaluated by Coomassie stained gels) of the GST- fusion proteins GST-p53, GST-PDZ1, GST-hDLG, GST-E6AP and GST-alone (as negative control) were incubated with 50 ng of E6 proteins from HPV16, HPV18, and HPV11 for 2 h at 4 °C, to determine any binding interactions.
After removing the supernatant, the resin was washed five times with PBS^– containing 0.25 % Nonidet-P 40. The assays were carried out three times. The purified His₆-E6 protein (20 ng) from HPV16, HPV18, and HPV11 was used as a positive control. The proteins were analyzed using SDS-PAGE and immunoblotting.

REP1^∆AD and the full-length CGA1 were synthesized by GENEWIZ (Nanjing, China). Recombinant Rep1^∆AD-His, as well as its variants, and GST-Cga1 was expressed in E. coli BL21.Cells were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16 °C overnight. Pellets were resuspended in 25 mM Tris-HCl pH 8.0, 500 mM NaCl and lysed by sonication. For purification of His-tagged proteins, lysates were applied to a Ni-NTA column (Qiagen),washed with 20 column bed volumes (CV) of resuspension buffer with 50 mM imidazole, and eluted with 5 CV of elution buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl and 250 mM imidazole). For GST pull down experiments, lysates were applied to Glutathione-Agarose (Sigma),washed with 20 CV of resuspension buffer with 1% Tween-20, and eluted with 5 CV of elution buffer (10 mM reduced glutathione [Sigma], 50 mM Tris-HCl, pH 9.0). Protein purity was determined via SDS–PAGE (Fig. S4D & S5C).

Recombinant AMA1 cytoplasmic tail protein (residues 567–622) with a thrombin-cleavable GST fusion (GST-AMA1_cyt), or an AMA1 Ser₆₁₀Ala point mutant of the same protein (GST-AMA1_cyt _S610A), was expressed in Escherichia coli BL21 and purified as described previously [23 (link)]. For NMR experiments, labelled protein was produced using E. coli grown in M9 medium containing ¹⁵N ammonium sulphate and ¹³C glucose as the sole nitrogen and carbon sources. To cleave the GST component, 100 μg GST-AMA1_cyt protein was treated with one unit of human alpha-thrombin (HTI) overnight at 18°C in 50 mM Tris-HCl, pH 8.2, and 2 mM CaCl₂. Following the digestion, glutathione agarose (Sigma) was used in excess to trap GST in solution and was later removed by centrifugation. The protein solution was then passed through a Superdex 75 HR 26/60 column equilibrated with 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl to remove thrombin and residual GST. For phosphorylation, 200 μg of AMA1_cyt in 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl was treated with 3.5 μg of mouse PKAc-α (Bioaffin GmbH & Co KG) in the presence of 2 mM ATP, 20 mM MgCl2, and 2 mM DTT and incubated overnight at 30°C. The protein solution was then passed through a Superdex 75 HR 26/60 column equilibrated in 20 mM Tris-HCl, pH 8.2, and 150 mM NaCl. This step completely removed PKAc-α.