Crystallization screens

The commercially available sparse matrix crystallization screens were ordered from Nextal, Montreal, now Qiagen, Hilden: Crystal Screen PEGS II Suites Qiagen, Hilden Crystal Screen AmSO₄ Suite Qiagen, Hilden Crystal Screen MPD Suite Qiagen, Hilden

Figure 8.1: Marker M12 and SeeBlue Plus2 from Invitrogen A: Unstained protein standard for accurate molecular weight estimation, 4-12 % Bis-Tris Gel, stained with Coomassie R-250. B: Prestained protein standard for easy and clear band identification, 4-12 % Bis-Tris Gel, SDS buffer (adapted from Invitrogen).

8.2 Protein biological methods

8.2.1 Determination of protein concentration after Gill

Protein concentration can be determined by a calculated theoretical extinction coefficient at 280 nm^[182] based on the primary sequence. The method can only be used for highly pure proteins because the absorption of impurities, especially aromatic amino acids like tyrosine, trytophane, phenylalanine and histidine and in minor degrees disulfidbridges disturbs the measurement.

Following the Lambert-Beer law (equation 8.1), the protein concentration can be calculated in mg/ml.

E₂₈₀ =₂₈₀·c[P rotein]·d (8.1) with

E₂₈₀ = Absorption at 280 nm

280 = Absorption coefficient at 280 nm c[Protein] = Concentration of the protein [mg/ml]

d = cuvette width [cm]

The theoretical extinction coefficient ofKlenTaqDNA polymerase at 280 nm, is 110380 (U/(M

·cm), which equals an absorption of 1.188 at 1 g/l.

8.2.2 Determination of protein concentration using Bradford assay

Protein concentration was usually measured using the Bradford assay^[183]. The measurement depends on a shift of the absorption of coomassie brilliant blue in an acidic environment from

465nm to 595nm caused by protein binding. The commercially available bradford assay (Bio-Rad) consist of coomassie brilliant blue dye, ethanol and phosphoric acid. This assay is compat-ible with interfering ions or reducing substances. For application, the commercially available Bradford reagent was diluted 1:5 with water and a standard curve with 0.1 mg/ml - 1.0 mg/ml BSA (Bovine serum albumin) was measured. Therefore, 10 µl probe was mixed with 1ml diluted Bradford reagent and incubated for 5min at RT. Resulting measurement points were plotted against the concentration and a calibration curve was built. Using this calibration curve, unknown protein concentration can be determined by mixing 10 µl sample to 1 ml Bradford reagent and measuring the absorption.

8.2.3 SDS polyacrylamide gelelectrophoresis

Discontinuous glycine SDS Polyacrylamide gelelectrophoresis (SDS-PAGE) was used for pro-tein size analysis and determination of purity and homogeneity. SDS (sodiumdodecylsulfate) is an anionic detergent and denatures most proteins completely. Additional reducing agents like β-mercaptoethanol cleave disulfide bridges inside the protein. Resulting SDS-protein complexes have a constant SDS/protein ratio, where the proteins get negatively charged proportional to their molecular weight. The influence of the netto charge of the native protein is marginal, be-cause it is completly covered by SDS. Therefore, the proteins behavior in the electrical field is independent on the amino acid sequence. Proteins were separated in an electrical field through the polyacrylamide matrix, which acts as a molecular sieve and separates the proteins after their stokes - radius. SDS-PAGE was done in a dual gel caster (Hoefer) following Laemmli, 1970^[184] with a 4 % stacking and 12.5 % resolving gel. The 15µl protein extract were mixed with 15µl 2x SDS-PAGE loading buffer (50 mM Tris pH 7.1, 2 % SDS, 50 % Glycerol, 5 % β-mercaptoethanol, 0.5 % bromphenolblue) and boiled at95^◦Cfor 5 min. 20µl were loaded on the SDS-PAGE gel and separated in a running buffer (25 mM Tris HCl, 192 mM Glycerol, 0.1 % SDS) for 10 min at 160 V followed by 35 min at 250 V. Staining was done either with coomassie brilliant blue or with silver staining. For the coomassie brilliant blue staining, the

Substance stacking gel 4 % resolving gel 12.5 %

30% Acrylamide 2.15 ml 0.325 ml

higher Tris — 0.625 ml

lower Tris 1.25 ml —

ddH2O 1.65 ml 1.55 ml

APS 37.5µl 17.5µl

TEMED 5.0µl 2.5µl

Table 8.8: Composition of 12.5 % SDS-PAGE.

% formalin, 1.6x10⁻⁵ % Na2S2O3). The development occurs using fresh developing solution, until band intensities are adequate (1-3min). To stop the development, 2.3 M citric acid are added. The stained gel can be dried after washing the gel for 30 min in water.

8.2.4 Western blot

The western blot (immunoblot) method^[186,187] was used to detect the His₆-tag of the protein and to verify the FactorXa protease digestion of the His6-Tag. Therefore, a gel electrophoresis (SDS PAGE, chapter 8.2.3) was used, to separate the proteins after their charge and size. As marker, SeeBluePlus2 Prestained Standard, Invitrogen was used. In the immunoblot device^R from BioRad, the proteins separated by SDS-PAGE were transferred electrophoretically from the gel to a PVDF-membrane (BioRad) using 25 mM Tris HCl pH 8.3, 192 mM glycine, 20

% (v/v) methanol as transfer buffer. The transfer was done at 100 V for 2 h or at 80 V over night, on a magnetic stirrer using an ice box for cooling. After transferring, the membrane was washed two times for 10 min with TBS (Tris-Buffered Saline: 10 mM Tris HCl pH 7.5, 150 mM NaCl), blocked for 1 h with blocking solution (3 % BSA in TBS), followed by a washing step two times for 10 min with TBSTT (20 mM Tris HCl pH 7.5, 500 mM NaCl, 0.05 % Tween20, 0.2 % Triton X100) and 10 min with TBS. The membrane was incubated for 1h in the first antibody solution (10 ml blocking solution plus 5µl penta His antibody), followed by washing two times for 10 min with TBSTT and one time for 10 min with TBS. Then the second antibody (10 ml blocking solution, 2µl AP-conjugated anti-mouse, Ig G) was applied for one additional hour, followed by washing 5 times for 10 min with TBSTT and one time for 10 min with TBS. Then the membrane was rinsed with AP buffer (100 mM Tris HCl pH 9.5, 100 mM NaCl, 10 mM MgCl₂) and incubated in staining solution (66µl NBT Stock (nitro tetrazolium blue chloride), 33µl BCIP stock (bromochloroindolyl phosphate), 10 ml AP buffer), until bands became visible. Staining was stopped with 3 % acetic acid.

8.3 Molecular biological methods

8.3.1 Quantification of DNA

Pure DNA can be quantified using the absorption maximum atλ= 260 nm (purin and pyrimidin bases absorb the UV-radiation from 250-270 nm). According to the Lambert-Beer rule, there is a linear relationship between the extinction and the concentration of the DNA. Different em-pirically found factors exist for the different nucleic acids. For single stranded oligonucleotides the following rule can be applied (8.2):

[mM] = (15.2·A+ 12.0·G+ 8.4·T + 7.1·C) (8.2) [mM] = millimolar extinction coefficient at 260 nm

A = Number of adenosins in the sequence G = Number of guanosins in the sequence T = Number of tymidins in the sequence C = Number of cytosins in the sequence

The concentration of the measured DNA containing solution can be calculated with equation 8.3:

c[mM] = Abs₂₆₀

[mM⁻¹] (8.3)

For dsDNA, an approximation equation can be used: Abs₂₆₀= 1 equals a concentration of 50 µg/ml dsDNA.

8.3.2 Native agarose gelelectrophoresis

Double stranded DNA from PCR was analyzed by a native agarose gelelectrophoresis with 0.8-2.5 % (w/v) agarose, depending on the DNA size. For a 1 % agarose gel, 40 ml 0.5 % TBE-buffer (Table 8.9) were mixed with 0.4 g agarose and boiled in a micro wave. After cooling down lower than 60^◦C, the solution was transferred to a gel device with a comb for the gel pockets. DNA samples were mixed with 1/3(v/v) of the loading buffer and loaded into the gel pockets. 1kb DNA ladder (New England Biolabs NEB N3232S), was used as marker. The gel was running at a current of 120 V for 30 min. After finishing, the gel was stained using 0.01

% (w/v) ethidiumbromide in 0.5 % TBE- buffer (Table 8.9) for 10 min and destained using 0.5

Table 8.9: TBE and Agarose gel loading buffer for the native agarose gelelectrophoresis.

8.3.3 Preparative denaturing polyacrylamide gelelectrophoresis (PAGE)

Preparative polyacrylamide gelelectrophoresis (PAGE) was used for higher purification of the commercial HPLC purified oligonucleotides prior crystallization. Therefore, the oligonucleotides were seperated by a 12 % preparative polyacrylamide gel with a thickness of 1.5 mm and 52 cm run length. 12 % polyacrylamide gel were prepared in 250 ml scale (120 ml PAGE buffer B, (Table 8.10) + 105 ml 8.3 M Urea + 25 ml PAGE buffer A, (Table 8.10)). Polymerisation was started by addition of 1.8 ml 10 % (w/v) APS-solution and 90µl TEMED. Gel solution was poured into a silanised gel rack and was polymerized for 60 min. Preparative gels were pre-warmed at 1200 V with 1x TBE as electrophoresis buffer (Table 8.9). Before loading, samples were mixed with 2 volumes PAGE loading buffer, incubated at95^◦Cfor 10 min and transferred on ice. Bromphenolblue was used as size marker and the separation was carried out at 90 mA for 3 h. After the electrophoresis step, the preparative gel was transferred to a TLC-plate (Thin layer chromatography plate) and the DNA was visualized using UV-shadowing at 256 nm. DNA was excised from the gel using a sterile scalpel. Excised gel pieces from the polyacrylamide gel

PAGE loading buffer PAGE buffer A

20 mM EDTA 10 x TBE

80 % Formamide 8.3 M Urea

10x TBE buffer PAGE buffer B

890 mM Tris HCl, pH 8.0 25 % Acrylamide solution

890 mM boric acid 8.3 M Urea

20 mM EDTA 2 % N,N’-Methylenbisacrylamide

Table 8.10: Buffers for the preparative denaturing polyacrylamide gelelectrophoresis (PAGE).

were crushed by forcing them through a syringe and collected in 2 ml sterile eppendorf tubes.

Water was added to the crushed gel pieces and they were incubated at55^◦Cover night to elute

the DNA. The mixture was filtrated after incubation through silanised glass-fibres. Resulting DNA was dried using a speedvac (Eppendorf) and further purified by ethanol precipitation, to get rid of residual salt and other impurities. Therefore, the gel purified dried DNA was dis-solved in 300 µl 0.3 M sodium acetate and subsequently precipitated with 3 volumes 96 % ethanol. Precipitation was carried out for 20 min at−80^◦C. Precipitated DNA samples were centrifuged for 1 h at 25000 g,0^◦C. The supernatant was discarded and the remaining pellet was washed twice with 1 ml precooled 70 % ethanol (−20^◦C) and dried in a speedvac. The resulting desalted and dried DNA pellet was dissolved in water for further experiments.