Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX

Aaron S. Mendez; Carolin Vogt; Jens Bohne; Britt A. Glaunsinger

SOX was then subcloned using restriction sites BamHI and SalI (New England BioLabs) into pFastBac HTD. This vector was modified to carry a GST affinity tag and PreScission protease cut site as described (31). All SOX mutants were generated using single primer site-directed mutagenesis (32). Sequences were validated using standard pGEX forward and reverse primers. Generation of viral bacmids and transfections were prepared as described in the Bac-to-BacÒ Baculovirus Expression System (Thermo Fisher Scientific) manual. After transfection, Sf9 cells (Thermo Fisher Scientific) were grown for 96 hr at 22 °C using SF-900 SMF media (Gibco) substituted with 5% fetal bovine serum (FBS) and 1% antibiotic antimycotic (AA). Supernatant was transferred to a 6-well tissue culture plate containing 1 ml of 2x10^6 cells/well. Cells were incubated for 96hr to generate Cleaved protein was concentrated to ~ 1 mL using Amicon filter concentrator membrane cut off 30 kDa (EMD Millipore), then loaded onto a HiLoad Superdex S200 pg gel filtration column (GE Healthcare Life Sciences). Protein elutions were concentrated using an Amicon concentrator described above to 5 mg/mL and aliquots were snap frozen in liquid N 2 and stored at -80 °C.