2.3.1 Bacterial strains
TheE. coliBL21-Gold(DE3) was used for heterologous overproduction of theKlenTaqM747K.
This expression system, harboring the KlenTaq M747K sequence in the pASK37plus vector, purchased from IBA was taken over from C. Gloeckner.[130] Between the N-terminal His6 -tag and the protein-coding sequence is a Factor Xa cleavage site in the pASK37plus. The vector contains a tet promoter inducible with anhydrotetracycline for cytoplasmic expression.
A second expression system was used inE. coliBL21-Gold(DE3) with the plasmid pGDR11 (a pQE31 (Qiagen) derivative harboring thelacIq gene[140] obtained from J. Nesper) with a N-terminal His6-tag. Expression of the pGDR11 is under T5- promoter control of the phage T5 and regulated through the lac promoter.
2.3.2 Expression and Purification
Heterologous expression inE. coliBL21-Gold(DE3) was done in 10 x 0.8 l scale Luria Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) with 100 µg/ml car-benicillin for pASK37plus or with 100 µg/ml ampicillin for the pGDR11. The medium was inoculated with 1:100 (v/v) overnight culture and cells were grown at 37◦Cin a shaking in-cubator at 220 rpm. After cell growth reached the mid-exponential phase (OD600= 0.5-0.8), protein expression was induced with anhydrotetracyclin (AHT) in the pASK37plus to an end-concentration of 200µM and with 1 mM IPTG in the pGDR11. After 4 h expression, the cells were harvested by centrifugation at 5000 x g for 10 min. Subsequently, pelleted cells were lysated for 30 min at 35◦C and 250 rpm using 30 ml lysisbuffer (10 mM Tris HCl pH 9.0, 300 mM NaCl, 2.5 mM MgCl2, 0.1 % Triton X100, 1 mM Benzamidin, 1 mM PMSF, 2 mg/ml lysozyme) per l cell culture. The lysate was heat denatured at 75◦Cfor 50min to precipitate heat labile enzymes. After cooling down to room temperature, the solution was ultracentrifuged at 50 000 x g for 1 h. The supernatant was filtered through a sterile filter (cut off size: 50µm)
Figure 2.3: SDS-PAGE of expression and Ni-purification ofKlenTaqM747K. Arrow indicates position of the purified protein. M: Marker M12;
1: before induction; 2: 1 h expression; 3: 3 h expression; 4: before induction; 5: 1 h expression; 6: 3 h expression; 7: after heat denaturation;
8: Supernatant of Ni-slurry 9: first Ni-slurry washing step; 10: second Ni-slurry washing step; 11: elution with 0.5 M imidazole.
from the Ni affinity purification was concentrated to 2 ml and applied to a pre-equilibrated (50 mM Tris HCl, 20 mM MgCl2, pH 9.0) gel filtration Superdex 200 (160 ml, GE Healthcare) to remove the imidazole and change the buffer. Pure protein elutes after 70 ml and the protein was concentrated to 7 mg/ml using centrifuge filtration devices (Vivaspin 50 kDa, Vivascience).
Expression could be optimized using vector pGDR11 (Chapter 9.3.2) instead of using the lower yield vector pASK37plus. In detail, out of 8 l cell culture, approximately 12 mg protein could be achieved with pGDR11 with the same purity as the 3 mg yield from pASK37plus.
2.3.3 Crystallization and data collection
Crystallization trials were carried out. Therefore, the protein was concentrated to 7 mg/ml and 20 mg/ml and crystallization screens were prepared using rational design based on the condition of the open 1KTQ[120] form. A screen was prepared using the published condition (100 mM Tris HCl, pH 9.0, 50 mM MgCl2, 6 % (w/v) PEG 3350) by varying pH from pH 8.2 to pH 9.5 (Fig. 2.4). First crystals were achieved with a protein concentration of 20 mg/ml in the hanging drop method using 100 mM Tris HCl, pH 9.5, 50 mM MgCl2, and 12 % PEG 3350 as reservoir (Fig. 2.4A). In fact, this condition is at the end of the screen so a second refined rational design
screen (Fig. 2.4B) was prepared with extended steps regarding PEG 3350 concentration and with different MgCl2 concentrations. In all conditions of the second screen appeared crystals after one week with a suitable size of 300 x 80 x 10 µm (Fig. 2.5B, C) in a hexagonal plate shape. Additionally, another condition from Li et al. 2001[141] for the tertiary form (0.1 M Hepes, pH 7.5, 20 mM MnCl2, 0.1 M Na-Acetate, 10 % (w/v) PEG 4000) was tested and a screen was prepared (Fig. 2.6A).
1 2 3 4 5 6
pH 8.2 pH 8.5 pH 8.7 pH 9.0 pH 9.3 pH 9.5
100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350
A
1 2 3 4 5 6
pH 9.4 pH 9.4 pH 9.5 pH 9.5 pH 9.6 pH 9.6
100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 20mM MgCl2 50mM MgCl2 20mM MgCl2 50mM MgCl2 20mM MgCl2 50mM MgCl2
12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2
12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2
12% PEG3350 16% PEG3350 12% PEG3350 16% PEG3350 12% PEG3350 16% PEG3350
B
Figure 2.4: Example of a rational design crystallization screen: [A] Initial and [B] refined crystal screen fromKlenTaqM747K. Crystals appeared in A in condition D6 and in all conditions in B.
Figure 2.5: Crystals fromKlenTaqM747K (crystal plate 072), A: first hit at 100 mM Tris HCl pH 9.5, 50 mM MgCl2, 12 % PEG 3350, Size:
160µm x 70µm, B: Refinement screen of condition A: Size: 800µm x 300µm, Condition: 100 mM Tris HCl, 20 mM MgCl2, 12 % PEG 3350, pH 9.6. Space group P21, 3.0 ˚A, a=93.1 ˚A, b=84.1 ˚A, c=256.1 ˚A;α=γ= 90.0˚ ,β = 98.8˚ , 6 monomers per ASU C: Different crystals of the refinement screen, cracked on the down left hand side.
To prevent the crystals from secondary radiation damage, cryocrystallography is used and therefore, the crystals were frozen in liquid nitrogen and measured at 100 K. To avoid water crystallization, which would lead to ice rings and damage the crystal lattice, different cryopro-tectans were tested:
Cryoconditions had to be established, where water at 100 K is in vitreous state so that it does not diffract the X-rays, while the protein crystal is still well ordered. This was tested by using a ro-tating anode generator (Schneider, Offenburg, Germany) equipped with a Proteum Pt135detector (Bruker AXS, AG Welte). Sequential soaking of the crystals with reservoir solutions containing increasing glycerol concentration was tried. In the optimized concentration, the concentration was increased in 5% (w/v) steps until 20 % (w/v) glycerol were reached. Different crystals of theKlenTaqM747K were measured either on beamline ID14-4, ID23-2 or ID29-1 on the ESRF in Grenoble, France or the beamline X06SA at the SLS in Villigen, Switzerland. In Figure 2.7, the diffraction pattern of one frame from theKlenTaq M747K is shown, which diffracted to 2.7 ˚A on the beamline ID29-1 ESRF, in Grenoble. Data reduction was done with XDS[142],