Protein analytical methods

2.3.1 SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Mini-PROTEAN® Tetra Cell Electrophoresis System (Bio-Rad Laboratories GmbH, München, Germany) according to the method of Schägger and Von Jagow, 1987 with minor modifications. The stock solutions prepared for SDS-PAGE gel electrophoresis are given in Table 10.

Table 10: Stock solutions for SDS-PAGE.

*no correction of pH required, **prepared with HPLC grade water

MATERIAL AND METHODS 35

Handcast gels (8.3 x 7.3 cm, 1 mm thickness) were prepared according to the pipetting scheme in Table 11. Separating gels were prepared by mixing acrylamide:bisacrylamide solution (29:1, 30% w/v, 3.3%

C; SERVA Electrophoresis GmbH, Heidelberg, Germany), gel buffer, deionized water, and 25% SDS solution (w/v). Gels were poured after the addition of fresh (<1 month) 10% ammonium persulfate (APS) solution and N,N,N’,N’-tetramethylethylendiamine (TEMED), overlaid with isopropanol, and allowed to polymerize. Subsequently, the isopropanol layer was discarded and a 4% stacking gel was prepared. The stacking gel was filled onto the separating gel, a comb was inserted to form sample cavities, and the gel was allowed to polymerize. The acrylamide solution, 10% APS, and TEMED were stored at 4 °C. All other solutions were kept at RT. For electrophoresis, the electrophoresis apparatus was assembled and running reagents were poured into the tank (anode buffer) and gel chamber (cathode buffer).

Sample preparation was done as following; the endolysins where thawed on ice and diluted to the desired concentration with the sample buffer. The samples were mixed 1:5 with the 5x loading buffer and denatured at ca. 95 °C for 5-10 minutes. Afterwards, 10 µL of each sample was loaded into a separate gel well. A molecular weight marker (1 µL) was loaded simultaneously with the samples in each run (PageRuler Plus Prestained Protein Ladder, 10-250 kDa; Thermo Fisher Scientific Inc., Rockford, lL USA). Electrophoresis commenced with 5 min at 80 V followed by a constant voltage of 100 V for ca. 90 minutes. After the electrophoretic run, gels were silver stained (2.3.3).

2.3.2 Acidic native PAGE

Acidic native PAGE was performed in the same gel electrophoresis system as used for SDS-PAGE.

Importantly, the polarity of the leads was reversed as the proteins will be positively charged at an acidic pH and run to the cathode (see isoelectric point in Table 16). The stock solutions prepared for acidic native PAGE gel electrophoresis are given in Table 12.

The separating and stacking gel were principally prepared in the same fashion as described for SDS-PAGE. The pipetting scheme of both separating and stacking gels is given in Table 13. The acrylamide solution, 10% APS, and TEMED were stored at 4 °C. All other solutions were kept at RT.

Table 11: Composition of separating and stacking SDS-PAGE gels.

Solution 12 % Separating gel 4% Stacking gel 30% Acrylamide

Table 12: Stock solutions for acidic native PAGE.

Solution Compound(s) Conc. pH

*no correction of pH required, **pH adjusted with 1 M KOH, ***prepared with HPLC grade water

36 MATERIAL AND METHODS

The acidic native PAGE loading buffer (5x) was prepared by mixing 1.45 mL 50% glycerol (v/v) and 0.5 mL separating gel buffer with traces of fuchsine as tracking dye. Aliquots of the loading buffer (5x) were kept at -21 °C. Sample preparation was performed on ice. Endolysins were thawed and diluted to the desired concentration with the stacking gel buffer, mixed 1:5 with the 5x loading buffer, and loaded immediately onto the gel. Electrophoresis started by 5 min at 20 A followed by a constant current of 40 A for ca. 120 minutes. After the electrophoretic run, gels were silver stained (2.3.3).

2.3.3 Silver staining

Proteins on acidic native PAGE and SDS-PAGE gels were silver stained according to the method of Blum et al., 1987 with modifications. The composition of reagents and procedure are given in Table 14. All steps were performed at RT on a platform shaker or by gentle manual shaking. Gels were scanned directly after completion of the staining procedure (Bio-5000 VIS Gel Scanner, SERVA Electrophoresis, Heidelberg, Germany).

Table 13: Composition of separating and stacking native PAGE gels.

Solution 10 % Separating gel 4% Stacking gel Separating gel buffer

Table 14: Silver staining protocol. Silver staining according to Blum et al., 1987 with modifications.

Step Reagent Duration

Silver impregnation 2 g/L silver nitrate (AgNO3) 20 min

Wash deionised water 3 ^x 20 sec

Development 30 g/L sodium carbonate (Na2CO3)

4 mg/L sodium thiosulfate pentahydrate (Na2S2O3 · 5H2O)

*Ethanol used was HPLC grade. **Formaldehyde was added to the developer shortly before used.

MATERIAL AND METHODS 37

2.3.4 UV-spectrophotometry for protein concentration and aggregation

Endolysin concentration and possible aggregation were determined by UV-spectrophotometry with a microscale spectrometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA).

2.3.4.1 Determination of endolysin concentration

The molar extinction coefficient at 280 nm (ε280) of Listeria phage endolysins was calculated based on the amino acid sequences using the following formula (Gill and Von Hippel, 1989):

ε₂₈₀ = ΣTrp × 5500 + ΣTyr × 1490 + ΣCysteine × 125

The specific attenuation coefficient (^0.1%A280) could subsequently be calculated with the molecular mass (Mr) of the endolysins as provided by the supplier:

0.1%A₂₈₀=ε₂₈₀ M_r

Protein concentrations could be determined by applying the Beer-Lambert law:

c = A₂₈₀

0.1%A₂₈₀× d

where c is the concentration (mg/mL), A280the measured absorption at 280 nm, and d the cuvette thickness (cm). Molecular mass and molar extinction coefficient are given in Table 15.

2.3.4.2 Protein aggregation

Determination of the ratio between the absorbance of a sample at 280 and 340 (i.e. absorption index) is a quick and easy method to measure the extent of protein aggregation. The peak of protein absorbance usually lies at 280 nm due to absorption of light by the aromatic side chains. Aggregated protein samples scatter light, causing the absorption measurement to display a hyperbole shape.

Typical absorbance spectra for precipitated and soluble forms of a protein are shown in Figure 7.

Table 15: Molar extinction coefficient and molecular mass of Listeria phage endolysins.

Endolysin Molecular extinction coefficient ε*

(M^-1 cm^-1)

Molecular mass Mr (kDa)

PlyP40 60,195 38.2

Ply511 56,060 36.5

PlyP825 54,235 34.2

Figure 7: UV-abosrbance of protein aggregates. The UV-absorbance spectrum of the precipitated (a) and soluble (b) form of BSA. Adapted with permission from Vincentelli et al., 2004. Copyright © 2004, John Wiley and Sons.

38 MATERIAL AND METHODS

2.3.5 Asymmetric flow field-flow fractionation

Asymmetric flow field-flow fractionation (AF-4) is a one-phase chromatography technique which allows for the separation of macromolecules based on colloidal size. Smaller particles are transported more rapidly along the channel than larger particles, which theoretically allows for the differentiation between protein monomers and aggregated protein complexes. As such, possible aggregation of endolysin PlyP825 was investigated by AF-4 (Wyatt Technology, Germany) coupled to multi-angle laser light scattering (MALS) (Dawn Heleos II, Wyatt Technology, Germany) and UV-detection (Dionex Ultimate 3000, Thermo Fisher Scientific, USA). Experimental procedure was performed under the supervision of Dr. F. Jakob (Lehrstuhl für Technische Mikrobiologie, Technische Universität München) as described in Jakob, 2014 and Ua-Arak et al., 2016 with minimal modifications. In short, endolysin PlyP825 was serially diluted with IPB and different concentrations (ranging from 5 to 200 µg/mL) were injected into the separation channel. The injection and elution flow were set to 1 and 5 mL/min, respectively. The gradient cross flow rate was kept constant at 5 mL/min. The separations were performed on 10 kDa regenerated cellulose membranes (Superon GmbH, Germany) with IPB (pH 6.2) as eluent solution. The data was collected and analysed using ASTRA 6.1 software (Wyatt Technology, Germany).