Expression and Purification with His 6 -tag

2.4 Results and discussion

3.3.1 Expression and Purification with His 6 -tag

Expression and purification of the His6-tag containingKlenTaqM747K was similar as described under 2.3.2.

Figure 3.2: Factor Xa digestion site in pASK-IBA37plus shown in blue.

natively carbenicillin) were inoculated from a frozen cell stock. 10 ml of the overnight culture were used for inoculating the 10 x 0.8 l medium flasks. Overnight cultures were grown at35^◦C and 150 rpm. The main culture was agitated by shaking in the cell incubator at 250rpm and 37^◦C. At OD600= 0.5 (normally about 2.5 h after inoculation) expression was induced with 100 µg/l anhydrotetracyclin. After a 4 h expression period, the cells were centrifuged at 20 000 rpm, for 15 min (typically about 9 g wet cell paste out of 8 l medium). The cells were resuspended in twice the cell weight TMN buffer (50 mM Tris-HCl pH 8.55, 10 mM MgCl2, 16 mM (NH₄)₂SO₄.) and were adjusted to a final concentration of 0.2 % Thesit and 0.2 % Triton X100. Cell lysis was done with lysozyme (end concentration: 5 mg/ml) while shaking for 30 min on ice and the resulting material was heat denaturated for 20 minutes at80^◦Cand cooled down to37^◦C. Protease inhibitor PMSF was added to an end concentration of 1 mM and the cell debris were centrifuged at4^◦Cfor 1 h at 35 000 rpm in the ultracentrifuge. To get rid of the E. coliDNA, a polyethylenimine (PEI) precipitation was carried out at high salt concentration by adding 1 ml 5 M NaCl to the solution to an end concentration of 0.25 M NaCl in 20 ml.

Precipitation was brought about by adding about 2 ml of 5 % (w/v) PEI dropwise under shak-ing on ice. The white precipitate was pelleted by centrifugation and the PEI precipitation was repeated, until no new precipitate was formed. The last centrifugation step was at 20 000 rpm for 30 min. During this critical step, it was necessary to add not too much PEI, otherwise the protein precipitated as well. This was checked by SDS PAGE of the precipitate. The resulting solution was diluted 1:1 with Factor Xa protease buffer (20 mM Tris-HCl pH 8.0, 0.1 M NaCl, 2 mM CaCl) and concentrated to 6 ml. Those 6 ml were dialyzed against Factor Xa protease buffer overnight. The buffer was changed and the dialysis continued for an additional 1 h. Then the dialyzed material was digested with 15µl Factor Xa protease for 48 h at room temperature.

After digestion, the resulting material was loaded on a 1 ml Ni-column (GE Healthcare). The

Ni-column was washed with 8 ml gel filtration buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol) the flow through and the 8 ml wash fractions were collected and used for the following gel filtration. Undigested protein was eluted and wasted using 500 mM imidazole in gel filtration buffer. For size exclusion chromatography, a 160ml Superdex75 column was equilibrated with the previously described gel filtration buffer. The concentrated Ni-column flow through and wash fractions were applied to the column and the resulting pure protein peak fraction was pooled and concentrated to 7 mg/ml.

3.3.3 Crystallization and data collection

The method used to obtain a ternary complex of the KlenTaq M747K was analogous to the strategy used by Li et al. (1999)^[64], Li et al. (2001)^[141] and first developed by Pelletier et al. (1994)^[157] for DNA polymerase β. Primer and template were annealed using standard procedures and the same buffer condition likeLi et al.(1998)^[124]. In the following, the annealed template and primer are designated as p/t. For crystallization with p/t and ddCTP, p/t oligomers were chosen following Liet. al(1998)^[124]:

Primer : 5’-GAC CAC GGC GC-3’

Template : 3’-CTG GTG CCG CGG GAA A-5’

+ ddCTP

The oligomers were ordered HPLC purified from MWG and later on from Purimex (no differ-ences in crystallization behavior between MWG and Purimex were observed) and used for crys-tallization. After several unsuccessful crystallization trials with DNA and ddCTP, the oligomers were purified further using native gelelectrophoresis (chapter 8.3.2). During native gelelec-trophoresis, no significant impurity of the oligomers could be detected and in the crystallization trials no distinct behavior was observed. It was concluded that the purity of the HPLC puri-fied oligomers was sufficient for crystallization. For the following experiments DNA oligomers were used directly, without purification by native gelelectrophoresis. The ternary complex was formed in presence of a final concentration of 20 mM MgCl₂ by mixing 3.0 mM DNA (p/t-complex) with 7-25 mg/ml protein and after 1h reaction time at room temperature, ddCTP was added in a molecular ratio of 1:7.5:50 (protein:DNA:ddCTP). Crystallization trials were done after an additional 1h reaction time at room temperature.

to seed crystals of the open form (Chapter 2), which had been grown in the hanging drop method against a reservoir containing 100 mM Tris-HCl pH 9.5, 50 mM MgCl2, 11 % (w/v) PEG 3350 (Fig. 3.3A) to a solution of 0.1M Hepes pH 7.5, 20 mM MnCl2, 0.1 M Na-acetate, 10 % PEG 4000 (Fig. 3.3B) containing 25 mg/ml protein, DNA and ddCTP (protein/DNA/ddCTP:

0.4 mM/3 mM/20 mM). Interestingly, crystals dissolved and recrystallized in a different space group (Fig. 3.3D in P3₁21 compared to Fig. 3.3A which is space group P2₁). Those small crystals (Fig. 3.3D) diffracted to 5 ˚A at the beamline X06SA at the Swiss Light Source (SLS) at the Paul Scherrer Institute in Switzerland. A reason for this recrystallization can be, that the

Figure 3.3: Seeding of crystals of the open form without DNA (A) to a solution with protein, DNA and ddCTP (B). After a few minutes (C), the crystals recrystallized in a different crystal form (D). The crystals diffracted to 5 ˚A and belonged to space group P3121 (in contrast to (A) which is space group P21).

protein concentration (25 mg/ml) or the precipitant concentration are too low causing a high protein solubility and the nucleation barrier cannot be overcome to build crystals. By seeding a protein crystal with roughly 700 mg/ml protein concentration in the crystal, the protein

con-centration in the hanging drop increases dramatically in this area. Another possibility is that the open form (which is assumed to be active in the crystal^[158]), binds DNA and ddCTP from the surrounding solution and reorientes to the closed conformation. These small crystals could be reproduced using the same reservoir condition and protein/DNA/ddCTP (0.4 mM/3 mM/20 mM) and a seed of the small crystal in the closed form in Figure 3.3D. However, the best crystals diffracted to 4.5 ˚A only, which is insufficient to see differences of the side chain conformation.

To improve crystal quality, the influence of the His6-tag on crystallization was tested. The low yield expression vector pASK37plus::KTQM747K has a Factor Xa cleavage site, to digest the His6-tag from the protein. Digestion of His6-tag fromKlenTaqM747K is shown in Figure 3.4.

Digestion of 1.1 mg protein in 2 ml Factor Xa-protease buffer and 5µl Factor Xa protease (1 mg/ml) needs 41 h to digest most of the His₆-tag from the protein (Figure 3.4B line 5). Shown on the SDS-PAGE (Fig. 3.4A) and the corresponding Western blot in Figure 3.4B. To separate undigested from digested protein, the mixture was applied to a Ni-column (Amersham) and the flow through and wash fractions were used for further size exclusion chromatography. Protein

Figure 3.4: Digestion of 1.1 mg protein in 2 ml Factor Xa protease buffer and 5µl Factor Xa protease (1 mg/ml). A: Coomasie stained SDS PAGE. B: Western blot of the same gel using anti-His antibody. 1: Marker; 2: before digestion; 3: after 17 h; 4: after 22 h; 5: after 41 h; 6:

after 65 h.

without His6-tag and gel filtrated, was concentrated to 25 mg/ml and crystallized in hanging drop (2+2 µl) with a protein:DNA:ddCTP ratio of 0.4 mM:3 mM:20 mM against a reservoir containing 0.1 M Hepes pH 7.3, 20 mM MnCl2, 0.1 M Na-acetate, 7 % (w/v) PEG 4000 (Fig.

3.5). Several crystals appeared and diffracted to 1.8 ˚A at the beamline X06SA at the SLS. Data reduction was done with XDS^[142]. Crystals belonged to the space group P3₁21 with cell axes of a=b= 107.9 ˚A, c= 89.9 ˚A andα=β= 90 ˚ ,γ= 120 ˚ . As the crystals were almost isomorphic to those of 3KTQ, the structure could be solved using 3KTQ (space group P3₁21, with a=b=

108.03 ˚A c= 90.17 ˚A andα=β= 90 ˚ ,γ= 120 ˚ ) as model for rigid body refinement. Refinement with Refmac^[149–153] led to a R_cryst=18.7 % and a R_{f ree}=24.1 %. Data collection statistics for theKlenTaqM747K bound to DNA and ddCTP (ternary complex) are shown in Table 3.1. The high resolution structure is shown in Figure 3.8. In Figure 3.8A, the overall structure is shown

Figure 3.5: Crystal ofKlenTaqM747K with DNA and ddCTP grown with the hanging drop method using 2µl+2µl protein/reservoir against a reservoir containing 0.1 M Hepes, 20 mM MnCl2, 0.1 M Na-acetate, 8-14 % PEG 4000, pH 7.1-7.6.

oriented like viewing the inner site of a slightly closed right hand which is bound to DNA and trapped with ddCTP. A side view 160^◦rotated to the right is shown in Figure 3.8B and the two catalytically active Mg²⁺-ions are shown as yellow spheres. One ddCTP was incorporated by theKlenTaqM747K and due to the lack of the 3’-OH group, the next ddCTP is in waiting po-sition prior to incorporation (can well be seen in Fig. 3.8D). The interaction of K747 with the p/t and the position of the ddCTP is schematically drawn in Figure 3.6. The first two adenines from the 5’-end of the 16mer template are not resolved in the density and are not built in the structure. Near the 3’-end of the primer in Figure 3.6 is the ddCTP trapped in the active center opposite a G and close to the incorporated ddCTP, dephosphorylated as ddC, in the adjacent base. Comparing the structure of theKlenTaqM747K with the KlenTaqwild-type shows that the orientation of the side chain does not change from methionine to lysine (Figure 3.9). Core rmsd betweenKlenTaqM747K andKlenTaqwild-type is 0.330 ˚A (rmsd was calculated using SSM Superposition^[159] implemented in Coot^[154]). The structure demonstrates, that the longer side chain is orientated beside the DNA and has no direct sterical effect to the backbone of the DNA. Therefore, the sterical properties of the longer side chain are probably not responsible for the different behavior regarding intrinsic properties of thisKlenTaqM747K. In Figure 3.7, the experimental electron density (2Fo-Fc) is shown at 1.8 ˚A resolution as a blue mesh at 1 sigma. The lysine 747 is labeled and hydrogen bonds a water (3.5 ˚A away) which on its part binds to the oxygen of the template phosphate of the ribose (3.6 ˚A away). Although 3.5 ˚A is near the upper limit for a normal water bridge, which is between 2.4 ˚A and 3.5 ˚A (calculated from donor to acceptor). This water bridge could strengthen interactions with the DNA. More-over, the increased positive potential during the hydrophobic to cationic exchange might also strengthen the binding of the negatively charged DNA. This has also been demonstrated by C.

Gloeckner^[130], who randomized the position 747. Mutations with substitutions at position 747 to anionic charged aspartate and glutamate as well as the polar uncharged asparagine and

cys-Data collection

Resolution range [ ˚A] 50-1.83 20-2.10

Outer shell [ ˚A] 1.83-1.98 2.10-2.20

R_meas[%]^∗ 6.2 (49.9) 18.9 (37.9)

Unique reflections 50017 36286^#

Total reflections 365757 697814^#

Mean(I/σ)^∗ 18.8 (2.5) 21.5 (4.5)

Completeness [%]^∗ 98.3 (50.0) 99.9 (99.9)

Space group P3121 P3121

Cell dimensions a,b,c [ ˚A] 107.9, 107.9, 89.9 108.6, 108.6, 90.4 Cell dimensionsα, β, γ[ ˚ ] 90.0, 90.0, 120.0 90.0, 90.0, 120.0

R_cryst[%] 18.7 18.3

Rf ree [%] 24.1 23.4

r.m.s. deviation bond length [ ˚A] 0.014 0.020 r.m.s. deviation bond angle [ ˚A] 2.102 2.310

Average B factors [ ˚A²] 35.0 34.0

Max. likelihood estimate for co-ordinate error [ ˚A]

0.106 0.135

Table 3.1: Data collection statistics forKlenTaqM747K in ternary complex with DNA and ddCTP and DNA with dCMeTP.^#2 Datasets were merged with XSCALE. Rmeas= redundancy independent R-factor. (For definition of Rmeassee Diederichs & Karplus (1997), Nature Struct.

Biol. 4, 269-275)^∗Values in parentheses correspond to those in the outer resolution shell.

teine and the nonpolar hydrophobic valine, phenylalanine and leucine were not able to extend the primer. The nonpolar hydrophobic methionine and alanine and the polar uncharged serine and tyrosine could extend the primer marginally whereas the uncharged glycine could build the full length product. Therefore, only the basic cationic arginine and lysine as well as the polar uncharged glutamine could extend the primer efficiently. This indicates that the cationic charge at this position enhances the binding to the negatively charged DNA. Probably the stronger binding of the template can stabilize the template in the p/t-bound complex and a mismatch can more easily be incorporated and extended. The resulting surface charge is shown in Figure 3.10 mapped on a colonny surface by using Adaptive Poisson-Boltzmann Solver (APBS)^[160]. The blue color represents the cationic potential while red represents the anionic potential. KlenTaq wild-type is shown in Figure 3.10A andKlenTaqM747K in Figure 3.10B.

Figure 3.6: Interaction of the lysine 747 with the primer and template DNA in theKlenTaqM747K complex. In the primer one ddC is incorporated and a second ddCTP is in waiting position (both in blue). The template interacts with the mutated lysine 747 via a water bridge between lysine 747 and the oxygen of the phosphate from the ribose. Arginine 728 interacts at this postion with the DNA, too.

Figure 3.7: Electron density (2Fo-Fc) fromKlenTaqM747K at 1.8 ˚A at position 747K is shown in blue mesh at 1σ. Lysine 747 is marked and 3.5 ˚A away is a water which interacts (3.4 and 3.6 ˚A away) with the two oxygens of the phosphate from the template DNA. 2.5 ˚A away from the first water is a second water which is also 3.4 ˚A and 3.6 ˚A away from the next two oxygens of the phosphate from the adjacent template DNA.

This water bridge network is only observed in theKlenTaqM747K and not visible in theKlenTaqwild-type. This interaction can stabilize the template at this position.

Figure 3.8: Structure ofKlenTaqM747K bound to p/t and ddCTP, p/t is shown in sticks, protein in cartoon. The two Mg²⁺-ions are colored as yellow spheres. The arrow indicates the position of the lysine 747 of the mutantKlenTaqM747K. A: View orientated like the open right hand. B: side view, rotated x= -160^◦, y= +10^◦from A. C: Zoom to the active site of A. D: Zoom to the active site of B. The ddCTP is visible coordinated by the two Mg²⁺-ions opposite the coding template and near the 3’-end of the primer. The difference of this mutant to the wild-type is the mutated lysine side chain which is indicated with a black arrow in Figure 3.8A-D, detailed in C.

Figure 3.9:KlenTaqM747K in blue is compared with theKlenTaqwild-type (3KTQ) in green. The position of the mutated side chain is indicated by a black arrow. Core rmsd betweenKlenTaqM747K andKlenTaqwild-type is 0.33 ˚A (rmsd was calculated using SSM Superposi-tion^[159]implemented in Coot^[154]).

Figure 3.10:KlenTaqwild-type (3KTQ) andKlenTaqM747K are shown in molecular surface representation with positive (blue) and negative (red) electrostatic potential. The p/t is drawn in sticks. Close-up views of the enzyme contacts with the sugar-phosphate backbone of the first base pair in the template strand are shown in A:KlenTaq(3KTQ) M747 is marked with a white arrow and is less cationic (less blue) than K747 in B fromKlenTaqM747K. Colonny surfaces were calculated with PyMol, electrostatic potential molecular surfaces with Adaptive Poisson-Boltzmann Solver (APBS)^[160]. B: The discussed additional water is represented as red sphere at the more cationic residue K747.

3.4.2 KlenTaq M747K in ternary complex with p/t, ddCTP and dCMeTP

Soaking experiments with dCMeTP and dCEtTP were done, to see how the sterically 4’-methyl- or 4’- ethyl- modified deoxycytosine triphosphates are incorporated and how they fit in the active center. To remove all bound ddCTP, crystals of theKlenTaqM747K tertiary form were soaked in the fresh reservoir solution and after one hour the surrounding reservoir solution was exchanged. After a period of 14 h, dCMeTP or dCEtTP, was added in high stoichiometric excess: 1µl, 1.5µl and 2µl of 20 mM dCMeTP or dCEtTP per 4µl protein drop. Only crystals with dCMeTP could be used, while crystals with dCEtTP cracked and were destroyed. Data collection statistics of theKlenTaqM747K bound to DNA with one ddCTP incorporated and a dCMeTP in waiting position, are shown in Table 3.1. In Figure 3.11A, the experimental 2Fo-Fc map is shown in blue, where dCMeTP is Watson-Crick base paired to guanosine. 3.8-4.3 ˚A be-low the dCMeTP is Phe667, which interacts through aromaticπ-πinteractions with dCMeTP.

Opposite to the Phe667 is the adjacent dideoxycytosine base π-interacting with the dCMeTP.

The binding site of dCMeTP is also known as the insertion site.[158,161–164]In Figure 3.11B, the 4’-methyl group and the 3’-OH group of the dCMeTP are visible and the density was clearly visible at this position at the reasonably high resolution of 2.1 ˚A. At the top in Figure 3.11B Glu615 is shown, which is oriented to the methyl group and is 3.4 ˚A away. The dCMeTP is shown in Figure 3.11C where two Mg²⁺-ions are shown in lime spheres and motif C, which interacts with the Mg²⁺-ions, is shown at the bottom (arrow). To show the space occupied by motif C from Figure 3.11C, it is shown in surface representation in Fig. 3.11D. During dephosphorylation, which is associated with a translation and rotation of the dCMeTP to the post-insertion site (Fig. 3.12), the 4’-methyl group of the ribose from dCMeTP may come into the vicinity of motif C. In the insertion site is enough space for dCMeTP as shown in Figure 3.12. But extension of this modified triphosphate can be difficult, because there is very less space for the 4’-methyl group in the post-insertion site. To understand how such triphosphate analoga fit into theKlenTaqbinding pocket, further studies of this behavior as well as kinetical measurements are in progress in the AG Marx.

Figure 3.11: Structure ofKlenTaqM747K bound to DNA and dCMeTP are shown. A: Measured 2Fo-Fc map is shown atσ=1.8 in blue. The dCMeTP is Watson-Crick base paired to guanosine (G). Phe667 is labeled and interacts through aromaticπ-πinteraction with the dCMeTP.

The adjacent incorporated ddC is shown at the top. B: Density for the methyl- and the 3’-OH- group of the dCMeTP is clearly visible at this 2.1 A high resolution. From the top Glu615 is shown, which is in the direction of the methyl group (3.4 ˚˚ A away). C: Side view of the dCMeTP, two Mg²⁺-ions are shown as green spheres. Motif C is shown from the bottom. The post-insertion site is marked as “p.i.”. D: Same orientation as in C, but with a zoom to the dCMeTP and motif C. Motif C is shown in surface representation. During dephosphorylation, which is associated with a translation and rotation of the dCMeTP to the post-insertion site (p.i.), the 4’-methyl group of the ribose from dCMeTP may come into the vicinity of motif C.

Figure 3.12: Structure ofKlenTaqM747K bound to DNA and dCMeTP is shown. The methyl group of the Watson-Crick base paired dCMeTP has enough space in the insertion site.

4.1 Abstract

KlenTaqM747K, I614K was crystallized in ternary form with ddCTP and primer/template. The well ordered crystals diffracted to 1.7 ˚A. Until now, this is the highest resolution of the Klenow fragment of Thermus aquaticus DNA polymerase I (KlenTaq), as the wild-type structure was solved at 2.3 ˚A.

4.2 Introduction

Two single hydrophobic/cationic exchange mutants ofKlenTaqhave been found to possess en-hanced lesion bypass ability: M747K found at the Marx group and I614K from Patelet al.^[165]. Isoleucine 614 is at a different position compared to methionine 747 and forms part of the hydrophobic pocket for binding the base and ribose portions of the incoming nucleotide. Klen-TaqI614K exhibits a 10-fold lower base misincorporation rate, as well as a high propensity to extend DNA mismatches. Furthermore, this mutant has lesion bypass activity on abasic sites (Fig. 1.5[1]) and vinyl chloride adduct ethenoA (Fig. 1.5[6])^[165]. The error rate is more than 20-fold higher relative to the wild-type (80x10⁻⁵ forKlenTaqI614K compared to 3.3x10⁻⁵ for the KlenTaq wild-type) and the enzyme efficiently catalyzes both transition and transversion errors. Mutation I614K is a part of the highly conserved motif A (amino acids 605-617, Fig.

4.1) near motif C at the active center. The higher error rate and the lower fidelity combined with the lesion bypass activity makes this I614K mutant interesting to combine with M747K.

Figure 4.1: Motif A and Q-helix sequence alignment. Sequence alignment of 20 different prokaryotic organisms shows highly conserved sequences in motif A and in the Q-helix. ThepolAgenes were taken from GenBank^TMand analyzed with NPS a(PBIL)^[166]. In red are isoleucine (I614 forThermus aquaticus) from motif A and methionine (M747 forThermus aquaticus) from the Q-helix (adapted from^[130]).

C. Gloecker from AG Marx, University Konstanz, combined these two mutants and analyzed the cumulative effects of this double mutantKlenTaqM747K, I614K. He found that the Klen-Taq M747K, I614K has a higher activity for the full length product by using damaged DNA (stabilized abasic site, 8-oxo-A or 8-oxo-G) compared to theKlenTaq M747K. Moreover, the KlenTaq M747K, I614K was able to amplify the full primer and add one additional base pair at the 3’-end independent of the template sequence. The incorporation of adenosine opposite an abasic site is known from the A-family polymerases as the A-rule.^[167,168] In the case of the KlenTaqM747K, I614K this property was more pronounced than in theKlenTaqwild-type. Ad-ditional differences exist in the processivity where the distributiveKlenTaqwild-type shows an

Im Dokument Intrinsic properties of a KlenTaq DNA polymerase mutant with enhanced substrate specificity determined by X-ray structure analysis (Seite 38-0)