Protein Biochemical Methods

3.2.2.1. Bradford Assay

The Bradford assay is a convenient and very fast method to determine protein concentrations in aqueous solutions. The procedure is performed according to the manufacturer’s instructions (Bio-Rad).

The assay is based on the fact that the absorbance maximum of the Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both, the hydrophobic and ionic interactions stabilize the anionic form of the dye, are causing the visible colour change.

To 20 µl of a protein solution are added 980 µl dilute Bio–Rad protein assay solution (diluted 1:5, v/v; 1 mg/ml Coomassie Brilliant Blue G 250 in 85% H3PO4). After 5 minutes incubation time the absorbance is measured at 595 nm and calibrated against a blank value, set up with ddH2O. The protein concentration is then determined by calibration against BSA standards. In parallel protein standards (1, 2, 3, 4, 5 and 6 mg/ml) are measured, which are then used to determine the concentration of the protein solution.

3.2.2.2. SDS-PAGE (SDS-Poly Acrylamide Gel Electrophoresis) The SDS-Poly Acrylamide Gel Electrophoresis was used to separate proteins by size and was performed according to (Laemmli, 1970). The purpose of this method is to separate proteins according to their size, independent of other physical feature. The solution of proteins to be analyzed is first mixed with SDS (Sodium dodecyl sulfate), an anionic detergent, which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass.

The denatured proteins are then applied to one end of a layer of polyacrylamide gel.

An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel. Depending on their size, each protein will move in a different way through the gel matrix.

Two glass plates were cleaned once with ddH2O and once with absolute ethanol, before the plates are used to assemble a gel chamber with the help of 1.2 mm spacers. The separating gel was cast using either 8 or 12% polyacrylamide gel solutions (see table below) to separate proteins in the molecular-weight range of interest. Polymerization was started by

addition of TEMED and 10% ammonium-persulfate. The gel was poured immediately up to

¾ of the height of the glass plates and the acrylamide- solution was overlaid with a few ml of H2O in order to prevent air-exposure and allow the acrylamide solution polymerize evenly.

The gel was allowed to polymerize for 1 hour at room temperature. After polymerization, the H2O covering the gel was removed and the stacking gel was prepared and poured immediately, a comb was inserted, and the gel was allowed to polymerize for 1 hour at room temperature. After complete polymerization of the polyacrylamide, the gel was associated with a gel chamber submerged with electrophoresis buffer. Protein samples are mixed with 100 µl SDS sample buffer and heated to 95°C for 10 minutes and 40 µl of each were loaded into a sample well of the gel. Proteins are focused while moving through the stacking gel at 15-25 mA and separated in the separating gel at 35 mA.

12% Separating

Separating Gel Buffer (4×) Stacking Gel Buffer (4×) 1.6 M Tris/HCl pH 8,8

0.4% (w/v) SDS

500 mM Tris/HCl pH 6.8 0.4% (w/v) SDS

Electrophoresis Buffer SDS Sample Buffer 192 mM Glycine

3.2.2.3. Staining of proteins with Coomassie Brilliant Blue

Coomassie Blue staining is based on the non-specific binding of the dye Coomassie Brilliant Blue R 250 and G250 to basic and aromatic amino acid side chains.

The gel is soaked in a solution of the dye for about 1 hour. During the subsequent destaining procedure, only the dye bound to the protein is retained in the gel. The gel is then fixed in the fixing solution for about 1 hour and dried. Coomassie Blue binds to proteins approximately stoichiometrical, so this staining method is useful when relative amounts of protein need to be determined by densitometry. The detection is limited to 0.1–0.5 µg of protein.

Staining solution Destaining solution 0.25% (w/v) Coomassie Brilliant Blue R 250 40% (v/v) Methanol 0.1% (w/v) Coomassie Brilliant Blue G 250 10% (v/v) Acetic acid 40% (v/v) Methanol

10% (v/v) Acetic acid Fixing solution

3% Glycerol (v/v)

30% Methanol

3.2.2.4. Affinity purification of MBP-fusion proteins

The hPrp31 cDNA was subcloned into pETM–41 (EMBL, Heidelberg) to produce MBP-fusion proteins. E. coli strain HMS174 was transformed with pETM–41–hPrp31 and grown at 20°C with shaking (200 rpm) to an OD600 of 0.8 in LB media in the presence of Kanamycin (see table below). IPTG was added to a concentration of 1 mM to induce protein expression. After further incubation for 3 hours at 20°C with shaking, cells were pelleted.

Cells were resuspended in 25 ml buffer B and incubated for 1 hour at 4°C in the presence of 1 mg/ml lysozyme (Sigma Aldrich) and one tablet of complete protease inhibitor (Roche, Mannheim). Cells were then subjected to sonication with a microtip (Branson) with three 20 second bursts at an amplitude of 40%. Insoluble material was pelleted by centrifugation at 10.000 g for 30 min, and the MBP–hPrp31–containing lysate was loaded directly onto 2 ml of Amylose Sepharose slurry (Amersham Pharmacia Biotech). Bound MBP–hPrp31 was washed once with 20 ml buffer C and twice with buffer B. Bound MBP–hPrp31 was eluted then with buffer B containing 10 mM maltose and finally fractions of 500 µl each (usually 3-4 fractions) were collected. Protein content of the fractions was detected by Bradford (see 3.2.2.1). The peak fractions were pooled and dialysed against buffer B.

Buffer B Buffer C Elution Buffer

Expressed proteins and transformed E.coli strains are listed:

Recombinant protein vector Resistance Strain for expression MBP-hPrp31 wt and

deletion mutants

pETM-41 Kanamycin E.coli HMS174

3.2.2.5. Affinity purification of GST–fusion proteins

The glutathione S-transferase (GST) affinity tag allows for rapid, near homogeneous purification of proteins expressed from GST expression systems. GST-15.5K and GST–

hPrp31 are expressed in E.coli and isolated from bacteria in a similar procedure as described above in section 3.2.2.4.

2 ml of GST resin are washed with 20 ml of buffer 2 in a 50 ml Falcon tube and the protein-rich supernatant from a 1 litre E.coli culture is added. GST resin and supernatant are incubated for 1 hour at room temperature shaking gently. Supernatant is removed after spinning down the beads at 2000 rmp and GST-sepharose washed two times with 20 ml buffer 2. During the last washing step, the slurry of beads is poured into an empty column (Bio-Rad) and the wash buffer is released by gravity flow. GST-fusion proteins can be released from the GST resin keeping the GST-tag. In this case, 6 ml of elution buffer are added to the GST resin and the eluate collected in 500 µl fractions by gravity flow.

Another possibility is to cleave the GST-tag while the protein is still bound to the GST-sepharose. The GST-tag remains on the beads while the protein is released to the supernatant. To perform this cleavage reaction, 6 ml of buffer 2 and 75U Thrombin or PreScission protease are added to the slurry and incubated over night shaking gently at room temperature. The supernatant is released by gravity flow from the empty column and collected in 500 µl fractions. The beads are washed two times with 2 ml buffer 2 and the wash is collected. Protein concentration is determined by Bradford (see 3.2.2.1).

To remove the Thrombin protease from the eluate after cleavage, 50 µl P-Aminobenzamidine Agarose (binding capacity 10-20 mg/ml), which binds the Thrombin protease are added, and incubated for 30 minutes at 30°C shaking gently.

P-Aminobenzamidine Agarose is removed from the eluate by centrifugation and filtration.

Without loss of activity, eluates can be stored at -80°C for prolonged periods of time.

Buffer 2 Elution Buffer 50 mM Tris pH 7.9

Recombinant protein vector Resistance Strain for expression GST-15.5K pGex–4–T–2 Ampicillin E.coli BL21 pLysS GST-hPrp31 pGex-6-P-1 Ampicillin E.coli BL21 RP