Primer stocks and dilutions were arrayed in 96 well V bottom microplates selleck inhibitor and stored at 20 C. Site directed mutagenesis was performed using the Quik Change kit using the plasmids pGEX4T 1 GST Inhibitors,Modulators,Libraries Thr TEV MK2 and pGEX4T 1 GST Thr TEV MK2 as templates. PCR was performed in 96 well V bottom microplates using a DNA Engine Dyad thermocycler complete with an ALS 1296, 96 well alpha unit. Reaction mixtures were cycled 18 times according to this schedule 95 C, 50 s. 60 C, 50 s. 68 C, 14 min. Cycling was preceded by incu bation at 95 C and followed by incubation at 68 C. Transformation of XL 10 Gold Ultracompetent E. coli cells was performed Inhibitors,Modulators,Libraries in 24 well RB Blocks. Thawed cells were mixed on ice with 2 L of into BL21 strain for expression studies. Competent cells were transformed with 0.
5 L of each plasmid in 24 well blocks as above. The transformation mix was spread onto 100 mm LB agar plates containing 100 g mL ampicillin and incu bated overnight at 37 C. Starter cultures grown Inhibitors,Modulators,Libraries at 37 C were used to inoculate 2. 5 mL of LB medium containing 100 g mL ampicillin in 24 deep well blocks. Once an OD600 of 0. 4 0. 6 was reached, the blocks were shifted to 18 C for 1 h and then induced with IPTG for 4 h. Cells were harvested by centrifugation, frozen at 80 C. Large scale cultures were induced in the same way with the exception that the flasks were shifted to 4 C for 40 min prior to induction. Analysis of the soluble and insoluble fractions was per formed by SDS PAGE on 4 20% gels. Cells were thawed and resuspended for lysis in Bug Buster HT. Bugbuster HT con taining 60 kU rLysozyme was added to the cell suspension.
The mixture was incubated on ice for 15 min before addition of 0. 2 mL of Bugbuster HT containing 20 mM DTT and Complete, EDTA free Pro tease Inhibitor cocktail. An aliquot of the crude lysate was separated by cen trifugation. the supernatant was analyzed as the soluble fraction. The pellet Inhibitors,Modulators,Libraries was resuspended in an equal volume of BugBuster HT and analyzed as the insoluble fraction. Proteins Inhibitors,Modulators,Libraries were visualized with SimplyBlue stain Western blotting was used to confirm protein identity. Gels were transferred to PVDF membranes using a Protean II Mini Trans Blot apparatus. After blocking membranes with 5% nonfat dry milk in TBS T buffer, the blot was probed with horseradish peroxidase conju gated anti GST antibody diluted 1 1000 in TBS T 5% non fat milk.
Membranes were washed extensively with TBS T, and then bound ATPase antibodies were visualized using the SuperSignal West Pico chemiluminescent substrate kit. Purification MK2 variants were purified using the following procedure at 4 C Cell pellets were thawed and resuspended in lysis buffer containing 50 mM TrisHCl, 250 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA, pH 7. 5. The cell suspen sion was sonicated on ice for 20 s iterations and then cen trifuged for 45 min.