smQTIKCVVVGDGAVGKTCLLISYTTNKF PSEYVPTVFDNYAVTVMIGGEPYTLGLFD TAGQEDYDRLRPLSYPQTDVFLVCFSVVS PSSFENVKEKWVPEITHHCPKTPFLLVGT QIDLRDDPSTIEKLAKNKQKPITPETAEK LARDLKAVKYVECSALTQKGLKNVFDEAI LAALEPPEPKKSRRCVLL
Expression: A number of colonies from the transformation were used to inoculate 80 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 4x 1L of TB medium (20 ml starter culture into each) containing 25 µg/ml kanamycin. When the OD600 reached ~1 the temperature was reduced to 25degC for 1 hour before induction with the addition of 1.0 mM IPTG. The expression was continued overnight (~ 16 hours). Cell harvest: Cells were spun at 4000 rpm for 10 mins at 4°C. Each 1L pellet was resuspended in 25 ml Resuspension Buffer. The resuspended cell pellets were placed in a -80°C freezer.
Nucleotide exchange procedure and TEV protease digestion: The eluted protein was concentrated to 40 mg/ml (0.5 ml volume). To this protein solution was added EDTA (to a final concentration of 20 mM - addition of 60 µl of 0.5M stock), 100 units of Calf intestinal alkaline phosphatase (CIP), DTT (to a final concentration of 1 mM), TEV protease (addition of 100 µl of homemade protease at ~2 mg/ml) and GppCp (addition of 200 µl of 10 mM stock). The solution was left at 4°C overnight.
Column 2: Gel filtration. Hiload S200 16/60 - 120 ml volume.
Procedure: The protein solution was loaded on the gel filtration column in GF buffer at a flow rate of 1.0 ml/min. Eluted proteins were collected in 1.75 ml fractions. The fractions containing protein were identified on a coomasie blue stained gel.
Rebinding of impurities to Ni-NTA: The protein was mixed with Ni-NTA resin (pre-equilibrated into GF buffer) at 4°C for 30 minutes. The resin was spun down and the supernatent collected.
Concentration: The nucleotide exchanged and TEV protease cleaved CDC42A was concentrated to 16 mg/ml, distributed into 50 µl aliquots and frozen at -80°C.