LA CIENCIA POLÍTICA Y SU RELACIÓN CON LAS DEMÁS CIENCIAS SOCIALES

The presence of a purification tag can sometimes influence the activity and stability of the protein of interest271–273. To avoid this, the tags were cleaved using protease digestion when an appropriate cleavage site was present (see section 7.1.5).

7.2.2.6.1 Cleavage by TEV digestion

The TEV protease is a cysteine protease that originates from the tobacco etch virus. It was expressed and purified as a maltose binding protein (MBP)-fusion protein with the mutation S219V by Volkmar Fieberg from the Famulok group. The protein contained a TEV cleavage site so it could be separated from MBP after self-cleavage. TEV protease was added to the protein of interest in a molar ratio of 1:50 (TEV protease:protein of interest) and the mixture was transferred to a dialysis tubing with a molecular weight cut off of 10 kDa. The tubing was incubated in 2 L dialysis buffer at 4 °C on a magnetic stirrer over night. During dialysis, imidazole/glutathione was removed from the buffer containing the protein of interest via dilution. TEV digestion was used during the purification of DrrA, Rab1 and Vav1 followed by reverse affinity chromatography (see section 7.2.2.5.5).

Table 21: Dialysis buffers for TEV digestions.

Buffer Composition

Dialysis Buffer DrrA1 50 mM Tris, pH 8

300 mM NaCl 20 mM Imidazole

Dialysis Buffer Rab1 50 mM Tris, pH 8

300 mM NaCl 20 mM Imidazole

Dialysis Buffer Vav1 50 mM Tris, pH 7.8

150 mM NaCl 7.2.2.6.2 Cleavage by Thrombin digestion

Thrombin is a serine protease that is commercially available and was bought at Calbiochem. 100 units of Thrombin were used for the purification yield from a 2 L culture and the mixture was transferred to a dialysis tubing with a molecular weight cut off of 10 kDa. Dialysis was performed as described in section 7.2.2.6.1. Thrombin digestion was used during the purification of Rac1 and Tiam1 followed by reverse affinity chromatography (see section 7.2.2.5.5).

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Table 22: Dialysis buffers for Thrombin digestions.

Buffer Composition

Dialysis Buffer Rac1 50 mM Tris, pH 7.8

300 mM NaCl Dialysis Buffer Tiam1DHPH 50 mM Tris, pH 7.8

150 mM NaCl 7.2.2.7 Size exclusion chromatography

Size exclusion chromatography was used to increase the purity of proteins after affinity chromatography. Proteins were separated according to their size since the porous column material allows small substances to enter its holes while large ones flow through faster without entering. A Superdex 200 HiLoad 16/600 pg column was attached to the Äkta FPLC system and equilibrated with two column volumes of filtered and degassed storage buffer using a flow rate of 1 mL/min. The 5 mL elution fraction from an affinity chromatography was loaded onto the column and the run was performed at 1 mL/min flow rate. The peaks visible in the UV monitoring at 280 nm were collected in 2 mL fractions. Aliquots of 30 µL were taken from each peak, loading buffer was added in a final concentration of 1 x and SDS- PAGE analysis was performed as described in section 7.2.1.1. Size exclusion chromatography was used during the purification of Rab1, Rab5a, Rabex-5GEF, Rac2 and Rin1C.

Table 23: Storage buffers for size exclusion chromatography.

Buffer Composition

Storage Buffer Rab5a 50 mM Tris, pH 7.8

100 mM NaCl 2 mM MgCl2

Storage Buffer Rabex-5GEF 50 mM Tris, pH 7.8

50 mM NaCl

Storage Buffer Rac2 50 mM Tris, pH 7.8

300 mM NaCl 2 mM MgCl2

Storage Buffer Rin1C 50 mM Tris, pH 7.8

300 mM NaCl 7.2.2.8 Handling of purified proteins

After purification the proteins had to be prepared for the experiments to follow. The protein concentration had to be determined and in most cases increased before proceeding.

7.2.2.8.1 Determination of protein concentration

The protein concentration could be determined either by a photometric approach or by using the Bradford Assay that provides a colorimetric readout.

7.2.2.8.1.1 Photometric determination of protein concentration

The protein concentration can be calculated from the absorbance at 280 nm based on Tryptophan, Tyrosine, Histidine, Cysteine and Phenylalanine residues inside the peptide

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chain. Every protein has a certain molar absorption coefficient (ε) that can be calculated as described by Pace et al. in 1995274. The protein concentration could then be calculated via the law of Lambert-Beer:

c l A₂₈₀ 

A280: Absorption at 280 nm, ε: molar absorption coefficient (M-1 cm-1), l: length of cuvette (cm) and c: protein

concentration (M)

The absorption has been measured using a spectrophotometer or a NanoDrop 2000. The buffer in which the protein of interest was dissolved was used as blank sample.

7.2.2.8.1.2 Determination of protein concentration via Bradford assay

The Bradford Assay is based on the absorption of Coomassie brilliant blue G-250 that binds to basic and aromatic amino acid residues275. The absorption of protein-bound dye could be measured at 595 nm. From that, the protein concentration could be estimated by comparing the measured absorption with that of a bovine serum albumin (BSA) standard of known concentrations (167 µg/mL – 3 mg/mL). The Protein Assay Dye Reagent Concentrate was diluted 1:5 with ddH2O and 150 µL of that were mixed with 2 µL of the protein sample or

lysate that was to be quantified. Lysates were diluted 1:10 before the concentration was measured. The mixture was transferred to a transparent 96 well plate and the absorbance was measured using the infinite M1000 pro plate reader.

7.2.2.8.2 Increasing the protein concentration and buffer exchange

After purification the protein concentration often had to be increased in order to use the protein in different assays later on. VivaSpin Turbo ultrafiltration spin columns were used for that. The molecular weight cut off (MWCO) of the column was chosen to be at least 50 % smaller than the molecular weight of the protein of interest. The concentrator was filled up with the protein sample and centrifuged at 3000 x g and 4 °C until the desired protein concentration was reached. For buffer exchange or desalting, the protein sample was concentrated until the desired concentration, then the column was filled up with the final buffer and the sample was concentrated again. After three such cycles about 99 % of the initial salt content was removed.