Celda N°6 de Residuos Industriales No Peligrosos

2.4.1. Expression of unlabelled proteins

Recombinant proteins were over expressed in BL21 (DE3) E. coli strains that were suitable for protein expression using Isopropyl-D-1-thiogalactopyranoside (IPTG) induction. A single colony expressing the recombinant gene of interest was picked for a small-scale pre-culture in 5 mL LB broth containing Amp and incubated overnight (37˚C at 180 RPM).

For large scale expression, 10 mL of the overnight small-scale pre-culture was used to inoculate 1 L of LB broth containing and incubated (37˚C at 180 RPM) until an OD600nm of 0.7-1.0 was reached. IPTG was added to each 1 L culture to a final

concentration of 1 mM and incubated (22˚C at 180 rpm for 3 hours). Cells were harvested by centrifugation (10,000 x g for 10 minutes) (Beckman Coulter - Avanti J-20 XPI). The supernatant was discarded, and the cell pellets were collected in a falcon tube and stored at -80˚C.

2.4.2. Expression of labelled proteins

PASTA 4 to 5 with (pCWT006) were over expressed in BL21 (DE3) E. coli strains using IPTG induction. A single colony expressing the recombinant gene of interest was picked for a small-scale pre-culture in 100 mL LB broth containing Amp and incubated overnight (37˚C at 180 RPM). For large scale expression, 10 mL of the overnight small- scale pre-culture was used to inoculate 2 x 1 L of LB broth containing 1 mL Amp and incubated (37˚C at 180 RPM) until an OD600nm 0.6 was reached. The 2 L cultures were

harvested by centrifugation (10,000 x g for 10 minutes, 4˚C) (Beckman Coulter - Avanti J- 20 XPI). To obtain high yield of the labelled protein, the cell condensation method was used (Sivashanmugam et al., 2009). The supernatant was discarded, and cells washed and resuspended in 200 mL M9 salts. Cells were centrifuged again (10,000 x g for 10 minutes, 4˚C) (Beckman Coulter - Avanti J-20 XPI). The supernatant was discarded and resuspended in 500 mL M9 media wither supplemented with 1 g 15_{N Ammonium chloride}

and 0.5 g 13_{C Glucose. Both cultures were pooled together and continued to incubate at}

25°C and 180 RPM for 1 hour to equilibrate. After 1 hour, cells were induced with 1 mM IPTG and allowed to express overnight. Cells were harvested by centrifugation (10,000 x

g for 10 minutes) (Beckman Coulter - Avanti J-20 XPI). The supernatant was discarded, and the cell pellets were collected in a falcon tube and stored at -80˚C.

2.4.3. Preparation of cell lysates

In preparation of cell lysates, frozen cell pellets were left to thaw. Buffer A was added to the to 30 mL in a falcon tube and a protease inhibitor tablet (ThermoFisher - 88666F) was added. The cells were sonicated on ice (20 seconds at 70%, x 5) using an ultra-sonicator (Bandelin Sonoplus) and then centrifuged (45 minutes at 50,000 x g) (Beckman Coulter - Avanti J-20 XPI) to remove the cell debris and produce a clear lysate and then stored on ice for immobilised metal ion affinity chromatography (IMAC). 2.4.4. Protein Purification

The basic methods describing the main purification techniques used in this study are summarised below.

2.4.4.1. Affinity Chromatography

Proteins with a poly-histidine (x6) affinity tag were purified by immobilised metal affinity chromatography (IMAC) using pre-packed HiTrap HP Nickel column (GE Healthcare) at room temperature unless stated otherwise. Columns are washed with 10 column volumes (CV) sterile water and equilibrated with a further 10 CV with Buffer A (Binding and equilibration buffer; see Table 2.3). Protein supernatant are loaded directly onto the column using a peristaltic pump. Columns were washed further with 10 CV Buffer A to remove un-bound protein. Columns were attached to an AKTA and protein was eluted using a gradient with Buffer B. 5 mL fractions were collected and protein elution was monitored by absorbance at 280 nm and occasionally also at 230 nm. Columns were washed with 10 CV of buffer containing 1 M imidazole, 10 CV sterile water and 10 CV 20% ethanol and stored at 4˚C. Columns were stripped, cleaned and recharged between different protein preparations following manufacturer protocols.

2.4.4.2. Size exclusion Chromatography

For highly concentrated proteins and large volumes, preparative size exclusion chromatography was used. Proteins were separated by size using wither a Superdex 200 (16/60), Superdex 200 (26/60) or Superdex 75 (16/60) on an AKTA Pure system at RT.

Columns were equilibrated with 1.5 CV of the required buffer and samples loaded on via a super-loop. Elution was with 1 CV buffer at 1 mL min-1 _{and 5 mL fractions were collected.}

Protein elusion was monitored at 280 nm and occasionally 230 nm. For analytical studies, size exclusion chromatography was performed using a Superdex 200 increase 10/300 GL column (GE Healthcare). Pre-equilibrated columns were initially subjected to calibration using a LMW and HMW calibration kit (GE Healthcare) in the chosen buffer to allow for molecular weight determination. 100 μL of protein was loaded onto the column using an injection loop and elution was at 0.5 mL min-1_.

2.4.4.3. Buffer Exchange and concentration

Buffer exchange was either initially through size exclusion chromatography, or with a VivaSpin centrifugal concentrator (Sartorius) with a molecular weight cut off either 5 or 10 kDa unless otherwise stated. Concentrators were centrifuged at 4˚C at 3,000 x g in a bench-top centrifuge (Eppendorf Centrifuge 5810R) and if buffer exchange was required, refilled with exchange buffer until a sufficient dilution factor was reached. Protein solutions were concentrated until the required volume was achieved and subsequently quantified.

2.4.4.4. Protein Purification of TEV protease

To remove the poly-histidine tag of recombinant proteins, TEV protease was prepared and used. Buffers used are listed in Table 2.3. Cell pellets of TEV were re- suspended in Buffer A on ice and passed through the cell disruptor three times (10,000 psi). The lysate was centrifuged at 14,000 RPM for 45 minutes. The supernatant was collected and loaded onto a 5 mL HiTrap HP Nickel column (GE Healthcare) pre- equilibrated with Buffer A using a peristaltic pump at 5 mL min-1_{. The column was loaded}

onto an AKTA and a gradient was performed from Buffer A to Buffer B from 0 to 100% respectively over 30 minutes at 2 mL min-1_{. Fractions were analysed on a 12% SDS-PAGE}

gel and fractions were pooled for dialysis overnight in dialysis buffer overnight at 4˚C. The protein concentration was measured using nanodrop at 8 mg mL-1 _{and stored at -80˚C.}

2.4.5. SDS-PAGE analysis

Proteins were separated and visualised under denaturing conditions by SDS- Poly- acrylamide Electrophoresis (SDS-PAGE) with Tris-glycine buffer with either a 12 or 15 %

acrylamide gels. Gels were prepared and run on the Mini- PROTEAN tetra system (BIORAD). 4.2 mL of the separating gel (1.75 mL distilled water, 2.5 mL 1.5 M Tris-HCl pH 8.8, 100 µL 10% SDS, 4.0 mL 30% acrylamide (Geneflow - 12-0066), 30 µL 10% ammonium persulfate (APS) (fresh), 30 µl tetram-ethylethylenediamine (TEMED) (Sigma Life Sciences - t9281) was added and overlaid with 100% ethanol whilst it set to remove the meniscus. The stacking gel (6.1 mL distilled water, 2.5 mL 0.5 M Tris-HCl pH 6.8, 100 µl 10% SDS, 1.3 mL 30% Acrylamide (Geneflow - 12-0066), 40 µl 10% ammonium persulfate (fresh), 40 µl TEMED (Sigma-T9281) was then added to the gel once the ethanol was removed and the comb was added. Protein samples for analysis were prepared in sample buffer (12% 1 M Tris- HCl pH 6.8, 20% glycerol, 0.4% SDS, 1% bromophenol blue, 10% β-mercaptoethanol). The protein samples and 10 µl of low molecular weight calibration ladder (Phosphorylase b, Bovine Serum Albumin, Ovalbumin, Carbonic anhydrase, Trypsin Inhibitor, β - Lactalbumin) (GE Healthcare - 17-0446-01) were heat denatured in a heat block (80˚C for 10 minutes). The cast gel was loaded onto the electrophoresis unit Mini-PROTEAN tetra system (BIORAD) and 15 µl of each sample was loaded into the respective well and gels were run in electrode buffer (25 mM Tris, 0.192 M Glycine, 0.1 % SDS, pH 8.3) for 45 minutes at 180 V. Development of SDS-PAGE gels were stained overnight with instant blue (Expedeon - ISB1L) then washed with water. Gels were imaged GelBox (Syngene) with a short-pass filter. Precast gels were occasionally used for analysis using either Criterion TGX Stain-Free gels, 4-20% gradient gels (BIORAD) or Mini-PROTEAN TGX Stain- Free gels, 4-20% (BIORAD).

2.4.6. Western Blots

E. faecalis cells were cultures in BHI broth containing 20 ng µL-1

anhydrotetracycline at 37°C with agitation until OD600nm = 0.6. After centrifugation (5000

x g, 10 min, 4°C) pellets were resuspended in SDS loading buffer and cells were then lysed by sonication. 20 µL of crude cell extract was analysed by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane using a BioRad Mini Transfer Blot. The Western membrane was blotted in 20% milk powder in PBS buffer for 1 hour. The blot was washed for 3 x 5 minutes in PBS. Detection of GFP fusions was performed with an anti-GFP-HRP goat polyclonal antibody (ThermoFisher – GF28R) (1/2000 in 10 mL PBS- tween) and left to incubate overnight. The blot was washed for 3 x 5 minutes in PBS. After

a further 1hour wash, blots were imaged by HRP chemiluminescence. Briefly a 1:1 ratio of the ECL substrate (BioRad) was mixed to a final volume of 10 mL. The mixed ECL substrate was added to the membrane for 5 minutes. After, excess ECL substrate was removed from the membrane and the membrane was placed in between two sheets of acetate. The membrane was imaged on a digital imager (Image Quant, GE).

2.4.7. Protein Quantification

To determine protein concentrations the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies) was used. Each purified protein construct was determined measuring the protein absorbance at 280 nm and the specific molecular weight and molar extinction coefficients of each protein construct.