Rosetta 2 de3 plyss

Plasmids were transformed into the BL21 (DE3) pLysS strain of E.coli (Merck Millipore) or Rosetta2 DE3 pLysS (Merck) and the transformed cells were cultured overnight at 37°C in Luria Bertani broth (Sigma Aldrich) supplemented with 30μg/ml kanamycin sulfate (Melford Laboratories Ltd). A volume of 20ml of this overnight starter culture was used to inoculate each of 1 litre of Terrific Broth (Sigma Aldrich) broth supplemented with 30μg/ml kanamycin sulfate. When the OD₆₀₀ reached approximately 0.8, protein expression was induced by the addition of IPTG (0.1mM, 0.5mM or 1mM) (Melford Laboratories Ltd) and expression was allowed to proceed either for 16 hours at 18°C, or 4 hours at 37°C. A volume of 40ml was sampled from the 1 litre cultures and cells were harvested by centrifugation at 3220xg for 15 minutes (Eppendorf 5810R centrifuage and A-4-62 rotor). The supernatant was discarded and the cell pellets were snap frozen in liquid nitrogen prior to storage at -80°C.

The plasmid expressing GST-tagged RAD9 C-terminal region (GST-RAD9CT) was described previously (24 (link)). The plasmids expressing GST-tagged p21 C-terminal region (GST-p21CT) was gifted from Dr Anindya Dutta (The University of Alabama at Birmingham) (48 (link)). The plasmids expressing its derivatives were constructed by site-directed mutagenesis using oligonucleotides listed in Table S1. The plasmids expressing GST-tagged RAD9 (residues 356–370) (GST-RAD9C15) or their derivatives were constructed by inserting the annealed oligonucleotides (Table S1) between the BamHI and XhoI sites of pGEX-6P-3 (Cytiva). These plasmids were introduced into an Escherichia coli strain, Rosetta 2(DE3)pLysS (Merck). The cells carrying each plasmid were grown in LB medium at 37 °C to OD600 = 0.6, followed by addition of final 0.2 mM IPTG and incubation at 25 °C overnight. The cells were collected, lysed by sonication in Lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 1 mg/ml of lysozyme, 1 mM PMSF, and 20 μg/ml leupeptin) and centrifuged at 7.2 × 10⁴g for 30 min at 4 °C. The supernatant was recovered and used for GST pull-down assay.

A rabbit polyclonal anti-Efg1 antiserum was generated using His₁₀-tagged Efg1 produced in E. coli. The EFG1 ORF (allele ORF19.8243) residing on a XhoI-BamHI fragment was subcloned into pET19b (Novagen), downstream of the T7 RNA polymerase promoter. The resulting plasmid encoded a His₁₀-Efg1 fusion but contained a single CUG codon (residue 449) that encodes serine in C. albicans but leucine in E. coli. This codon was changed to a UCG serine codon by site-directed mutagenesis using oligonucleotides pET19Serinhin/her, resulting in plasmid pET19-His-Efg1Kodon, which was transformed into E. coli Rosetta 2 (DE3)pLysS (Merck). Transformants were grown and the T7 promoter was induced according to instructions of the manufacturer. Cells were resuspended in buffer (20 mM CAPSO pH 9.5, 1 M NaCl, 1 mM EDTA, 20 mM imidazol, 0.1% Triton X100) and broken using 3 passages through a French press cell (Slaminco Spectronic Instruments). Crude extracts were cleared by centrifugation and applied to HisTrap columns connected to an ÄKTA prime plus fraction collector (GE Healthcare). The His₁₀-Efg1 fusion protein was eluted using CAPSO buffer containing 250 mM imidazol. Purified protein (100 μg) was injected on days 1, 14, 28 and 56 in 2 New Zealand White rabbits (performed by Eurogentec, Belgium). One rabbit generated high anti-Efg1 titers in ELISA tests and in immunoblottings (dilution 1:5000).