3.4 Recombinant protein purification
3.4.2 Protein solubility screening
Throughout this thesis different solubility screens were performed. As a starting point protein-expressing cells were harvested and aliquots of 12 ml were decanted in 15 ml reaction tubes.
Afterwards cells were pelletized for 10 min at 3000 x g and 4 °C and stored at -20 °C. For cell lysis, the pellet was resuspended in a specific lysis buffers library 195, or in custom designed buffer palettes (Table 23-26) and transferred to 1.5 ml sonication-compatible reaction tubes.
Subsequently cell walls were disrupted using 1 mg ml-1 lysozyme for 1 h at 4 °C and sonication.
Sonication was performed using a Bioruptor® Pico (Diagenode, Seraing, Belgium) for 5 cycles with 30 s on and 30 s off. Cell debris was pelleted by centrifuging the cell lysate at 14000 x g at 4 °C for 30 min. Pellet and supernatant were analyzed by SDS-PAGE.
Table 23
Initial buffers, which were used for a GST-AtKRP125b solubility screen
No. Buffer pH Ions Detergent Additives
1 100 mM Tris-HCl 7.0 200 mM NaCl 0.1 M Urea 1 mM DTT, 1 mM ATP 2 100 mM Tris-HCl 7.0 50 mM NaCl 0.1 M Urea 1 mM DTT, 1 mM ATP 3 100 mM Tris-HCl 7.0 50 mM NaCl 0.1 % Triton 1 mM DTT, 1 mM ATP
4 100 mM Tris-HCl 7.0 10 mM NaCl 1 mM DTT, 1 mM ATP
5 100 mM Tris-HCl 8.0 200 mM NaCl 0.1 M Urea 1 mM DTT, 1 mM ATP 6 100 mM Tris-HCl 8.0 50 mM NaCl 0.1 M Urea 1 mM DTT, 1 mM ATP 7 100 mM Tris-HCl 8.0 50 mM NaCl 0.1 %Triton 1 mM DTT, 1 mM ATP
8 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT, 1 mM ATP
9 50 mM Pipes 7.0 1 mM MgSO4 1 mM DTT, 1 mM ATP,
1 mM EGTA
10 50 mM Pipes 7.0 1 mM MgSO4 0.1 M Urea 1 mM DTT, 1 mM ATP, 1 mM EGTA
11 50 mM Pipes 7.0 1 mM MgSO4 0.1 %Triton 1 mM DTT, 1 mM ATP, 1 mM EGTA
12 10 mM HEPES 7.2 1 mM MgCl2 25 mM NaCl
1 mM DTT, 1 mM ATP, 1 mM EGTA
13 10 mM HEPES 7.2 1 mM MgCl2
25 mM NaCl 0.1 M Urea 1 mM DTT, 1 mM ATP, 1 mM EGTA
Table 23 continued
No. Buffer pH Ions Detergent Additives
14 10 mM HEPES 7.2 1 mM MgCl2 25 mM NaCl
0.1 %Triton 1 mM DTT, 1 mM ATP, 1 mM EGTA
15 2x PBS 1 mM DTT, 1 mM ATP
16 2x PBS 0.1 %Triton 1 mM DTT, 1 mM ATP
Table 24
Different buffers, with different lysis conditions, which were used for a GST-AtKRP125b solubility screen
No. Buffer pH Ions Additives Cell Lysis
1 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT,
1 mM ATP 1 mg ml-1 Lysozyme for 15 min
2 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT,
1 mM ATP Sonication 3 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT,
1 mM ATP
1 mg ml-1 Lysozyme for 15 min, Sonication 4 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT,
1 mM ATP 1 mg ml-1 Lysozyme for 15 min, Sonication, 1 mM PMSF
5 100 mM Tris-HCl 8.0 10 mM NaCl 1 mM DTT, 1 mM ATP, 1 mM EGTA
1 mg ml-1 Lysozyme for 15 min, 1 mM PMSF 6 10 mM HEPES 7.2 1 mM MgCl2
25 mM NaCl 1 mM DTT, 1 mM ATP, 1 mM EGTA
1 mg ml-1 Lysozyme for 15 min
7 10 mM HEPES 7.2 1 mM MgCl2
25 mM NaCl 1 mM DTT, 1 mM ATP, 1 mM EGTA
Sonication
8 10 mM HEPES 7.2 1 mM MgCl2
25 mM NaCl 1 mM DTT, 1 mM ATP, 1 mM EGTA
1 mg ml-1 Lysozyme for 15 min
9 10 mM HEPES 7.2 1 mM MgCl2
25 mM NaCl 1 mM DTT, 1 mM ATP, 1 mM EGTA
1 mg ml-1 Lysozyme for 15 min, 1 mM PMSF
Table 25
Conditions, which were used to screen for a buffer in which GST-CHS1 appears to be soluble.
No. Buffer pH Ions Detergent Additives
1 100 mM Tris-HCl 7.5 150 mM NaCl 1 mM DTT
2 100 mM Tris-HCl 8.0 150 mM NaCl 1 mM DTT
3 100 mM PIPES-NaOH 7.5 150 mM NaCl 1 mM DTT
4 100 mM Tris-HCl 7.5 150 mM NaCl 1 mM DTT, 5 % Glycerol
5 100 mM Tris-HCl 8.0 150 mM NaCl 1 mM DTT, 5 % Glycerol
6 100 mM PIPES-NaOH 7.0 150 mM NaCl 1 mM DTT, 5 % Glycerol 7 100 mM PIPES-NaOH 7.5 150 mM NaCl 1 mM DTT, 5 % Glycerol
Table 25 continued
No. Buffer pH Ions Detergent Additives
8 100 mM Tris-HCl 7.5 150 mM KCl 1 mM DTT, 5 % Glycerol
9 100 mM Tris-HCl 8.0 150 mM KCl 1 mM DTT, 5 % Glycerol
10 100 mM PIPES-NaOH 7.0 150 mM KCl 1 mM DTT, 5 % Glycerol 11 100 mM PIPES-NaOH 7.5 150 mM KCl 1 mM DTT, 5 % Glycerol 12 100 mM Tris-HCl 7.5 150 mM LiCl 1 mM DTT, 5 % Glycerol 13 100 mM Tris-HCl 8.0 150 mM LiCl 1 mM DTT, 5 % Glycerol 14 100 mM PIPES-NaOH 7.0 150 mM LiCl 1 mM DTT, 5 % Glycerol 15 100 mM PIPES-NaOH 7.5 150 mM LiCl 1 mM DTT, 5 % Glycerol 16 100 mM Tris-HCl 7.5 150 mM NaCl 1 % Triton
X-100 1 mM DTT, 5 % Glycerol 17 100 mM Tris-HCl 8.0 150 mM NaCOOH 1 mM DTT, 5 % Glycerol
Table 26
Different buffers which were used for a GST-BON1 solubility screen 194.
No. Buffer pH Ions Detergent Additives
1 50 mM Tris-HCl 7.5 50 mM NaCl 5 mM EDTA
2 50 mM Tris-HCl 7.5 50 mM NaCl 500 mM Urea 5 mM EDTA 3
4 50 mM NaOAc 5.0 50 mM NaCl 5 mM EDTA
5 50 mM Tris-HCl 7.0 50 mM NaCl 5 mM EDTA
6 50 mM Tris-HCl 9.0 50 mM NaCl 5 mM EDTA
7 50 mM Tris-HCl 7.5 100 mM NaCl 5 mM EDTA
8 50 mM Tris-HCl 7.5 200 mM NaCl 5 mM EDTA
9 50 mM Tris-HCl 7.5 50 mM KCl 5 mM EDTA
10 20 mM Tris-HCl 7.5 50 mM NaCl 2.5 % Glycerol
11 250 mM Tris-HCl 8.0 50 mM KCl 0.2 % Triton
X-100 1 mM EDTA, 1 mM DTT, 1 mM AEBSF
12 10 mM Tris-HCl 7.4 150 mM NaCl
13 50 mM Tris-HCl 7.5 100 mM NaCl 5 mM EDTA, 1 mM
AEBSF
14 50 mM Tris-HCl 7.5 300 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF
15 50 mM Tris-HCl 7.5 200 mM NaCl 300 mM Urea 5 mM EDTA, 0.1 mM AEBSF
16 50 mM Tris-HCl 7.5 200 mM NaCl 100 mM Urea 5 mM EDTA, 0.1 mM AEBSF
17 100 mM Tris-HCl 7.5 200 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF
18 250 mM Tris-HCl 7.5 150 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF
19 50 mM Tris-HCl 7.5 200 mM NaCl 100 mM Urea 5 mM EDTA, 0.1 mM AEBSF
20 50 mM Tris-HCl 7.8 200 mM NaCl 300 mM Urea 5 mM EDTA, 0.1 mM AEBSF
Table 26 continued
No. Buffer pH Ions Detergent Additives
21 100 mM Tris-HCl 7.8 200 mM NaCl 300 mM Urea 5 mM EDTA, 0.1 mM AEBSF
22 200 mM Tris-HCl 7.8 150 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF
23 20 mM Tris-HCl 7.8 100 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF
24 150 mM Tris-HCl 7.8 150 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF 25 20 mM HEPES 7.8 150 mM NaCl,
2 mM MgCl2
0.1 mM AEBSF 26 50 mM HEPES 7.5 150 mM NaCl,
2 mM MgCl2
0.1 mM AEBSF
27 50 mM HEPES 7.5 200 mM NaCl 5 mM EDTA
28 100 mM HEPES 7.5 200 mM NaCl 5 mM EDTA, 0.1 mM
AEBSF 29 20 mM HEPES 7.8 150 mM NaCl,
2 mM MgCl2
0.1 mM AEBSF
30 50 mM HEPES 7.8 20 mM NaCl 5 mM EDTA