Expression of GST-fusion proteins

Materials

*

• LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,

ad 1l A. bidest)

• Ampicillin (100 µg/ml)
• IPTG (100 mM Isopropylthiogalactosid)
• 2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v)-β-
• Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) glycerin
• T-buffer 100mM Tris (pH 8.0), 5 mM EDTA
• GST-antibody

Procedure

Test expression:

After cloning, the constructs of the desired GST-fusion protein are transformed in E.coli BL21. Eight bacterial clones are selected for overexpression analysis. For the overexpression, bacterial clones are incubated in 5 ml antibiotica containing LB- medium for

6 h at 30°C and 220 rpm. After 6 h, 1ml of the culture is centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer. The remaining culture is induced with 0.1 mM IPTG and incubated overnight at 30°C and 220 rpm. At the next day

0.5 ml of each sample is centrifuged for 1 min at 13000 rpm, the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft he protein is analysed by SDS-Page and Coomassie-staining.

Expression for purification:

One bacterial clone is incubated in 30 ml antibiotica containing LB-medium overnight at 30°C and 220 rpm. After 12-18h, 300 ml antibiotic containing LB-medium is inoculated with OD₆₀₀=0,2 of the overnight-culture and incubated at 30°C and 220 rpm. At OD₆₀₀ = 0.6-0.8 1 ml is removed (sample before induction) and the remaining culture is induced with 0.5 mM IPTG for 3 h at 30°C and 220 rpm. Afterwards 0.5 ml is removed (sample after incuction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft the protein is analysed by SDS-Page and Coomassie-staining. The remaining cells are centrifuged for 15 min at 4700 rpm, washed with T-buffer and stored at -80°C.